皮膚感作性試験代替法 Local Lymph Node Assay (LLNA):
BrdU-ELISA

目次
評価会議報告書 ------------------------------------------------------------------------------

1

第三者評価報告書 ---------------------------------------------------------------------------

7

OECD GUIDELINE FOR THE TESTING OF CHEMICALS No.442B,
Skin Sensitization: Local Lymph Node Assay:
BrdU-ELISA---------------------------------------------------------------------------------------

15

ICCVAM Test Method Evaluation Report on the Murine Local Lymph Node Assay:
BrdU-ELISA A Nonradioactive Alternative Test Method to Assess the Allergic
Contact Dermatitis Potential of Chemicals andProducts--------------------------------------------------------------------------------------------- 31（財）化学物質評価研究機構より提案のあった皮膚感作性試験代替法(LLNA-BrdU 法)の一次評価報告書------------------------------------------------------------------ 65（財）化学物質評価研究機構より提案のあった皮膚感作性試験代替法(LLNA-BrdU 法)の二次評価報告書------------------------------------------------------------------ 81

皮膚感作性試験代替法 Local Lymph Node Assay (LLNA): BrdU-ELISAの判定基準の変更に関する評価会議報告

JaCVAM 評価会議

平成 24 年（2012 年）10 月 1 日

1JaCVAM 評価会議
吉田武美（日本毒性学会）：座長
浅野哲秀（日本環境変異原学会）
五十嵐良明（国立医薬品食品衛生研究所）
大島健幸（日本化学工業協会）
小笠原弘道（独立行政法人

医薬品医療機器総合機構）

小野寺博志（独立行政法人

医薬品医療機器総合機構）

黒澤 努（日本動物実験代替法学会）
杉山真理子（日本化粧品工業連合会）
西川秋佳（国立医薬品食品衛生研究所 安全性生物試験研究センター）
長谷川隆一（独立行政法人
牧

製品評価技術基盤機構）

栄二（日本免疫毒性学会）

増田光輝（座長推薦）
横関博雄（日本皮膚アレルギー・接触皮膚炎学会）
吉田 緑 （国立医薬品食品衛生研究所 安全性生物試験研究センター）
吉村 功（座長推薦）
渡部一人（日本製薬工業協会）
任期：平成 24 年 4 月 1 日～平成 26 年 3 月 31 日

以上

2

皮膚感作性試験代替法 LLNA: BrdU-ELISA については、既に JaCVAM 評価会議でその妥当性が評価されている 1)。今回 LLNA: BrdU-ELISA の判定基準の変更に関する感作性試験評価委員会からの報告 2)を受け、以下の 10 項目について評価したので報告する。
＜審議内容＞
1.

当該試験法は、どのような従来試験法を代替するものか。または、どのような毒性を評価ある
いは予測するものか。
当該 Local Lymph Node Assay (LLNA): BrdU-ELISA は、化学物質等の皮膚感作性を評価するモ

ルモット Maximization Test (GPMT)あるいは Buehler Test (BT)の代替試験法であるマウス LLNAの改良試験法である。従って、当該試験法の予測するところは、従来試験法の LLNA が予測する化
学物質等の皮膚感作性である。
2.

当該試験法と従来試験法の間にどのような科学的なつながりがあるか。
従来試験法は、感作に基づく耳介リンパ節の細胞増殖反応を放射性物質の[3H-methyl]-thymidine(3H-TdR)の DNA への取込みを指標として検出する試験法である。当該試験法は、感作誘導によるリンパ節細胞の増殖を検出するという試験法の原理は従来試験法と同じであるが、放射性物質の3H-TdR の代わりに、bromodeoxyuridine (BrdU)の DNA への取込みを指標とし、BrdU 量を酵素免疫測定法（ELISA）により吸光度として計測するものである。
3.

当該試験法とそのデータは、透明で独立な科学的評価を受けているか。
ICCVAM は、第三者評価委員会を組織し、JaCVAM が実施した当該試験法の検証試験 3)で得られた 10 物質の試験成績を含む 43 物質の試験成績をレトロスペクティブに解析し、併せて従来試験法による結果と比較した。その解析において、当該試験法における皮膚感作性の判定基準としてカット
オフ値＞1.6 を使用することにより、従来試験法と同等の結果が得られることを示し、当該試験法の精度、感度および特異性を評価した 4)。その組織および評価結果は、ICCVAM のホームページで公表されている。
また、我が国の LLNA: BrdU-ELISA 感作性試験評価委員会は、JaCVAM で実施された当該試験法の検証報告と上記 ICCVAM の評価結果を対比して評価した。
よって、当該試験法の判定基準の変更は、透明で独立な評価を受けていると判断される。
4.

当該試験法は、従来試験法の代替法として、どのような物質又は製品を評価することを目的としているか。
当該試験法は、皮膚外用剤として用いる医薬品、医療機器、化粧品、皮膚適用の医薬部外品、農薬等に含まれる物質又はそれらの製品に求められる皮膚感作性を評価する従来試験法の代替法としての使用を目的とする。
5.

当該試験法は、ハザード評価あるいはリスク評価のどちらに有用であるか。
当該試験法は、上記の物質又は製品における皮膚感作性のハザード評価に有用である。

3

6.

当該試験法は、目的とする物質又は製品の毒性を評価できるか。その場合、当該試験法の適用条件が明確になっているか。
当該試験法の妥当性を示すデータは、JaCVAM が検証試験に使用した 10 物質を含む 43 物質（化粧品、化成品、農薬、医薬品、殺菌消毒剤、合成中間体および原材料、食品添加物、香料、衛生材料
および溶剤）の試験成績である。よって、当該試験法は、これらを包括する物質又は製品の皮膚感作性を評価することができるといえる。
当該試験法においては、従来試験法と同様に過度の局所刺激性や明らかな全身毒性を示さない用量を最高用量とする。偽陰性を排除するため、皮膚感作性の陽性判定基準を JaCVAM で検証したカッ
トオフ値＞2.0 から＞1.6 に変更した。この変更された判定基準においては、偽陽性を示す物質も存在するため、皮膚感作性陽性の最終判定は、被験物質に関する付加的情報（例えば、用量反応情報、
全身毒性若しくは過剰な局所皮膚刺激性の証拠、タンパク結合性、分子量、関連化学物質の成績等）
を考慮して行う必要がある。
適用限界は、従来試験法の LLNA と同様である。
7.

当該試験法はプロトコルの微細な変更に対して頑健であるか。
当該試験法は、従来試験法と原理的に同じであることから、当該試験法の精度並びに施設内および施設間再現性および頑健性は、従来試験法と同じであると考えられる。当該試験法は、BrdU 測定に
市販の ELISA キットを使用することから、操作の変更（例えば、プレートの洗浄および乾燥、二次抗体の反応時間等）は測定値が変動する要因になりうる。しかし、実施に当たっては、ICCVAM の報告書を参照して施設毎に試験プロトコルを確立し、そのプロトコルを忠実に守ることが必要である。
8.

当該試験法の技術習得は、適切な訓練と経験を経ている担当者にとって容易なものであるか。
試験法の実施に特殊な設備が必要か。
当該試験法は、適切な訓練を受け、経験を積んだ担当者にとってその技術習得は容易である。従来試験法に比べ、放射性取扱施設等の特殊な設備は必要ない。
9.

当該試験法は、従来試験法と比べて時間的経費的に優れているか。
従来試験法は、リンパ球の増殖反応を測定する方法として放射性物質（RI）を使用するため、特殊な実験施設や設備を必要とし、放射能管理、廃棄物の処理問題等、試験を実施する上で種々の制約があった。一方、当該試験法は、通常の実験設備が使用でき、また、RI を必要としないことより、
特殊設備や廃棄物処理の管理が不要であり、時間的経費的に優れている。
10. 当該試験法は、動物福祉の観点及び科学的見地から、目的とする物質又は製品の毒性を評価する代替法として、行政上利用することは可能か。
当該試験法は、動物を用いない代替試験法ではない。しかし、GPMT 等他の皮膚感作性試験法と比較して、動物に与えるストレスは少なく、苦痛の軽減という点で優れている。皮膚外用剤として用いる医薬品、医療機器、化粧品、皮膚適用の医薬部外品、農薬等に含まれる物質又はそれらの製品の皮膚感作性を予測する当該試験法は、判定基準のカットオフ値を下げることにより偽陰性の排除が可能になるとともに、RI を使用せずとも従来試験法と同等の結果が得られることから、行政上利用することは可能である。
4

参考文献
1)

皮膚感作性試験（LLNA：BrdU 法）の評価会議報告書、JaCVAM 評価会議（平成 22 年 5 月、
平成 23 年 4 月改定）

2)

皮膚感作性試験代替法 Local Lymph Node Assay (LLNA): BrdU-ELISA の第三者評価報告（平成 24 年 7 月）

3)Hajime Kojima, et al. Inter-laboratory validation of the modified murine local lymph node
assay based on 5-bromo-2’-deoxyuridine incorporation. J. Appl. Toxicol. 31: 63-74 (2011)4)

ICCVAM (2010). ICCVAM Test Method Evaluation Report on the Murine Local Lymph
Node Assay: BrdU-ELISA, a Nonradioactive Alternative Test Method to Assess the Allergic
Contact Dermatitis Potential of Chemicals and Products. NIH Publication No. 10-7552.
Research Triangle Park, NC: National Institute of Environmental Health Sciences.

5

Local Lymph Node Assay: BrdU-ELISA (LLNA: BrdU-ELISA、
局所リンパ節試験: BrdU-ELISA)の概要およびその調査結果

平成 24 年 6 月 25 日

JaCVAM 皮膚感作性試験第三者評価委員会
委員長

金澤 由基子 [(独) 医薬品医療機器総合機構]

委員

牧 栄二 [前 (財) 食品農医薬品安全性評価センター]1要旨
マウスにおける Local Lymph Node Assay (LLNA，局所リンパ節試験)は、感作に基づく
耳介リンパ節の細胞増殖反応を放射性物質の[3H-methyl]-thymidine（3H-TdR）の取り込み量を測定することで定量的かつ客観的に判定する試験法である。LLNA: BrdU-ELISA は、
放射性物質の 3H-TdR の代わりに、 bromodeoxyuridine (BrdU)の取り込み量を酵素免疫測
定法により吸光度として測定し、細胞増殖の指標にしたものであり、原法の LLNA と同じ原理による試験法である。本試験法の原理並び簡便性は、海外においても認められるところであるが、被験物質の皮膚感作性陽性を判断するカットオフ値 [溶媒処置群（陰性対照群）
に対する被験物質処置群の BrdU の取込量の比(Stimulation index、SI 値)] に本邦と海外において違いが生じている。
本報告では、JaCVAM で実施された LLNA:BrdU-ELISA の検証報告（2008）とICCVAM の LLNA：BrdU-ELISA Evaluation Report (2010) を対比し、両者のカットオ
フ値（SI 値）の違いについて調査を行った。その結果、ICCVAM が検証の対象とした皮膚
感作性陽性 32 物質全てを陽性と判定するカットオフ値 1.6 を判定基準として採用することが妥当であると考えた。また、LLNA：BrdU-ELISA に関する OECD ガイドライン（2010）においても、皮膚感作性陽性を示すカットオフ値として 1.6 が設定されている。このような状況を踏まえると、本邦においても 1.6 をカットオフ値として設定することが妥当であると考える。一方、SI 値が 1.6 から 2 の間には、皮膚感作性偽陽性を示す化合物も存在するため、化合物の皮膚感作性の最終判定においては、ICCVAM が勧告する付加的な情報（例えば、用量反応情報、全身毒性若しくは過剰な局所皮膚刺激の証拠、必要に応じて、処置群と溶媒対照群の統計的な比較、ペプチド反応性、分子量、関連物質の結果、他の試験デー
タ）を考慮して決定する必要がある。
はじめにマウスにおける Local Lymph Node Assay (LLNA，局所リンパ節試験)は、皮膚外用剤として用いる医薬品ならびに化粧品原料を含む化学物質等の皮膚接触感作性のリスクを動物で予測するモルモットにおける Maximization Test (GPMT)或いは Buehler Test (BT)の代替試験法であり、その予測率は、GPMT に劣らないとされ 1)、国際的に認知されている。
LLNA は、感作に基づく耳介リンパ節の細胞増殖反応を放射性物質の[3H-methyl]-thymidine（3H-TdR）の取り込み量を測定することで定量的かつ客観的に判定する試験法である。LLNA: BrdU-ELISA は、放射性物質の 3H-TdR の代わりに、
bromodeoxyuridine (BrdU)の取り込み量を酵素免疫測定法により吸光度として測定し、細胞増殖の指標にしたものであり、原法の LLNA と同じ原理による試験法である。本試験法の原理並び簡便性は、海外においても認められるところであるが、被験物質の皮膚感作性のカットオフ値 [溶媒処置群（陰性対照群）に対する被験物質処置群の BrdU の取込量の比
2

(Stimulation index、SI 値)] に本邦と海外において違いが生じている。
2)と ICCVAM
本報告では、
JaCVAM で実施された LLNA: BrdU-ELISA の検証報告（2011）の LLNA: BrdU-ELISA Evaluation Report (2010) 3)を対比し、両者のカットオフ値の違い
について調査を行ったので、その結果並びに JaCVAM 皮膚感作性試験第三者評価委員会（以下、委員会）としての提案を述べる。
１． 試験法LLNA: BrdU-ELISA（図1）の試験法の原理は、原法のLLNAと同じである。LLNA:
BrdU-ELISAでは、LLNA同様に3日間連続して被験物質を耳介に経皮投与し、5日目に
3H-TdRの静脈内投与の代わりにBrdUを腹腔内投与し、6日目に採取した耳介リンパ節の感作に伴う細胞増殖を検出する。即ち、皮膚感作性を有する低分子化合物が経皮投与されると、皮膚組織中のタンパク質と結合し、感作抗原として皮膚の樹状細胞に認識される。
その後、樹状細胞は活性化しながら皮膚から所属リンパ節へ遊走し、抗原提示を行い、抗原特異的なTリンパ球細胞の増殖を誘導する。この一連の生体応答が感作誘導期である。
LLNAでは、感作誘導期のリンパ節における抗原特異的なTリンパ球細胞の増殖を放射性物
質の3H-TdRのDNAへの取り込みを指標として検出するが、LLNA: BrdU-ELISAでは、
BrdUのDNAへの取込を指標とし、BrdU量をEnzyme Linked Immuno Solvent Assay（ELISA）により検出するものである。
図１ LLNA: BrdU-ELISA の概略

２．JaCVAM の LLNA: BrdU-ELISA 検証試験における SI 値
JaCVAM の行った検証試験では、
7 施設が参加し、10 化合物 (2,4-dinitrochlorobenzene、
eugenol、formaldehyde、glutaraldehyde、hexylcinnamic aldehyde、isopropanol、lactic
acid、methyl salicylate、nickel sulfate、および trans-cinnamic aldehyde) について盲検下にて試験された。
検証試験では、3 化合物（isopropanol、2,4-dinitrochlorobenzene および hexylcinnamic
aldehyde）が全施設で検証され、7 化合物（eugenol、formaldehyde、glutaraldehyde、lactic3

acid、methyl salicylate、nickel sulfate、および trans-cinnamic aldehyde）が 3 施設で検証された。試験結果は、各々の化合物処置群について抗 BrdU 抗体を用いた ELISA 法による吸光度として示され、各々の化合物処置群の SI 値が、同時に測定された溶媒対照群に対する BrdU の取込量の比として求められた。その結果、皮膚感作性陽性を示す SI 値は、＞2 と設定された。
この検討で 10 化合物について得られた結果は、それら化合物の SI 値において施設間バ
ラツキは小さく、一貫性のあるものであった。SI 値＞2 における LLNA: BrdU-ELISA のGPMT/BT に対する感度、特異性および精度は、各々7/7 (100%)、3/3 (100%)および 10/10(100%) であった。
３．ICCVAM の LLNA: BrdU-ELISA Evaluation Report における SI 値（図 2 参照）
ICCVAM は、次のように結論している。即ち、LLNA: BrdU-ELISA の精度および信頼性は、化合物を、潜在的に皮膚感作性を有する物質或いは非感作性物質として検出するためには十分であり、皮膚感作性試験として LLNA: BrdU-ELISA を支持するものである。
JaCVAM の検証試験で得られた 10 化合物のデータを含む 43 化合物の検証データベースのretrospective 解析において、LLNA: BrdU-ELISA は、LLNA で皮膚感作性物質と評価された 32 化合物全て（0％[0/32]の偽陰性）と LLNA で非感作性物質と評価された 11 化合物の内 9 化合物（18％[2/11]の偽陽性）を正確に検出した。ICCVAM は、潜在的に皮膚感作性を有する化合物を検出する判定基準としてカットオフ値 1.6 を使用することを勧告した。
ICCVAM のこの勧告は、SI 値＞1.6 を使用する場合、原法の LLNA による最新の検証デー
タベースに対して偽陰性が生じないことに基づくものである。
図2

LLNA: BrdU-ELISA の SI 値と原法 LLNA の SI 値の比較

4

（化合物名の横の括弧内の数字は、LLNA: BrdU-ELISAに続いて原法のLLNAの試験数を示す。試験数
は、類似の試験の最高用量のみが記載されているので、得られた試験の総数とは異なる場合がある。）

LLNA: BrdU-ELISAの欠点として、1.6から1.9の間のSI値で陽性反応が得られる場合、偽陽性の結果を生じる可能性のあることが挙げられている。ICCVAMは、この点について
次のように報告している。即ち、皮膚感作性物質がLLNA:BrdU-ELISAの精度に干渉する
ある種の特性を持たない限り、LLNA:BrdU-ELISAの適用領域は原法のLLNAと同じ筈である。1つの例外は、nickel化合物である。原法のLLNAと異なり、LLNA:BrdU-ELISAは、
それらを潜在的な皮膚感作物質として正確に同定する能力を有し、nickel化合物の皮膚感作性を評価できる。
LLNA:BrdU-ELISA の精度は、原法の LLNA の精度に匹敵するものであった。最適な
LLNA:BrdU-ELISA の遂行は、皮膚感作性物質と非感作性物質を分類するためにカットオ
フ値 1.6 を使用することにより達成された。原法の LLNA と比較すると、精度は、偽陽性率 18% (2/11)、偽陰性率 0% (0/32)の 95％ (41/43）であった。LLNA:BrdU-ELISA において SI 値＞1.6 を使用すると、1.6 から 1.9 の間の SI 値を示す 2 偽陽性化合物（hexane および lactic acid）が生じた。それ故、1.6 から 1.9 の境界域の SI 値を示した化合物が潜在的に皮膚感作性を有する化合物であるか否かを確認するために、他に利用できる情報、例えば、
用量反応性、全身毒性あるいは過度な局所刺激性の証拠、必要に応じて SI 値と共に統計的有意性を考慮すべきである。また、その考察には、既知の皮膚感作性物質との構造類似性も含め、被験物質の種々の性質をも加えるべきであるとしている。

5

４．ICCVAM の勧告
最終的に ICCVAM は、次のように勧告している。即ち、カットオフ値 1.6 という一つの判定基準を潜在的な皮膚感作性物質を分類するために使用すべきである、何故なら、この基準が使用されるとき、原法の LLNA による最新の検証データベースにおいて偽陰性は認
められなかったからである。しかしながら、判定基準としてカットオフ値 1.6 を使用すると、原法の LLNA と比較して 18% (2/11)の偽陽性が生じる。LLNA:BrdU-ELISA において 2
偽陽性物質が 1.6 から 1.9 の間の SI 値を示したことから、この範囲での成績については、
真に陽性であることを確認するために付加的な情報（例えば、用量反応情報、全身毒性若
しくは過剰な局所皮膚刺激の証拠、必要に応じて処置群と溶媒対照群の統計的な比較、ペプチド反応性、分子量、関連物質の結果、他の試験データ）を考慮すべきである。
５．委員会としての提案
表 1 ICCVAM と JaCVAM の LLNA: BrdU-ELISA の検証の比較
LLNA: BrdU-ELISA
研究組織

検証に使用された

陽性判定の SI 値

化合物数
ICCVAM

43＞ 1.6

JaCVAM

10＞ 2

JaCVAM と ICCVAM の判定基準のカットオフ値の違いは、検証に使用した化合物数の違いも一因と考える。両者には検証に使用した化合物数に差があり、化合物数を多くすれば試験の精度は高まるが、皮膚感作性の判定基準であるカットオフ値は低くなることが予想される（表 1 参照）。
ICCVAM の検証において、検証の対象とされた 32 皮膚感作性物質の内 SI 値＜2 の皮膚感作性物質は、
僅か 2 化合物（2-mercaptobenzothiazole [MBT]および imidazolidinyl urea）
である。また、ICCVAM の基準である SI 値＞1.6 においては、11 非感作性物質の内偽陽性を示す 2 化合物（hexane および lactic acid）が存在することも事実である。
以上の結果を総合的に判断し、委員会としては、ICCVAM が検証の対象とした皮膚感作性陽性物質全てを陽性と判定できるカットオフ値 1.6 を判定基準として採用することが妥当であると考える。また、LLNA: BrdU-ELISA に関する OECD ガイドライン（2010）4)においても、皮膚感作性陽性を示すカットオフ値として 1.6 が設定されている。このような状況を踏まえると、本邦においても 1.6 をカットオフ値として設定することが妥当であると考える。一方、SI 値＞1.6 においては、皮膚感作性偽陽性を示す化合物も存在するため、化合物の皮膚感作性の最終判定においては、ICCVAM が勧告する付加的な情報（例えば、用量反応情報、全身毒性若しくは過剰な局所皮膚刺激の証拠、必要に応じて、処置群と溶媒
6

対照群の統計的な比較、ペプチド反応性、分子量、関連物質の結果、他の試験データ）を考慮して決定する必要がある。
6．文献
1) ICCVAM and NICEATM. The Murine Local Lymph Node Assay: A Test Method forAssessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds. The
Results of an Independent Peer Review Evaluation Coordinated by ICCVAM and
NICEATM. NIH Publication No. 99-4494. February (1999)
2) Hajime Kojima, et al. Inter-laboratory validation of the modified murine local lymph
node assay based on 5-bromo-2’-deoxyuridine incorporation. J. Appl. Toxicol. 31:
63-74 (2011)
3) ICCVAM (2010). ICCVAM Test Method Evaluation Report on the Murine Local
Lymph Node Assay: BrdU-ELISA, a Nonradioactive Alternative Test Method toAssess the Allergic Contact Dermatitis Potential of Chemicals and Products. NIH
Publication No. 10-7552. Research Triangle Park, NC: National Institute ofEnvironmental Health Sciences.
4) OECD Guideline for the Testing of Chemicals: Skin Sensitization: Local Lymph Node
Assay: BrdU-ELISA (TG 442B), 22 July (2010)7

OECD/OCDE

442B
Adopted:22 July 2010

OECD GUIDELINE FOR THE TESTING OF CHEMICALSSkin Sensitization: Local Lymph Node Assay: BrdU-ELISAINTRODUCTION
1. OECD Guidelines for the Testing of Chemicals are periodically reviewed in light of scientific progress,changing regulatory needs, and animal welfare considerations. The first Test Guideline (TG) for thedetermination of skin sensitization in the mouse, the Local Lymph Node Assay (LLNA; TG 429) was
adopted in 2002, and has since then been revised (1). The details of the validation of the LLNA and areview of the associated work have been published (2) (3) (4) (5) (6) (7) (8) (9). In the LLNA,
radioisotopic thymidine or iodine is used to measure lymphocyte proliferation and therefore the assay has
limited use in regions where the acquisition, use, or disposal of radioactivity is problematic. The LLNA:
BrdU-ELISA [Enzyme-Linked Immunosorbent Assay] is a non-radioactive modification to the LLNA test
method, which utilises non-radiolabelled 5-bromo-2-deoxyuridine (BrdU) (Chemical Abstracts Service[CAS] No 59-14-3) in an ELISA-based test system to measure lymphocyte proliferation. The LLNA:
BrdU-ELISA has been validated and reviewed and recommended by an international independent scientificpeer review panel as considered useful for identifying skin sensitizing and non-sensitizing test substances,with certain limitations (10) (11) (12). This Test Guideline is designed for assessing skin sensitization
potential of chemicals in animals. TG 406 utilises guinea pig tests, notably the guinea pig maximisation
test and the Buehler test (13). The LLNA (TG 429) and the two non-radioactive modifications, LLNA:
BrdU-ELISA (TG 442 B) and LLNA: DA (TG 442 A), all provide an advantage over the guinea pig tests
in TG 406 (13) in terms of reduction and refinement of animal use.
2.
Similar to the LLNA, the LLNA: BrdU-ELISA studies the induction phase of skin sensitization andprovides quantitative data suitable for dose-response assessment. Furthermore, an ability to detect skinsensitizers without the necessity for using a radiolabel for DNA eliminates the potential for occupational
exposure to radioactivity and waste disposal issues. This in turn may allow for the increased use of mice todetect skin sensitizers, which could further reduce the use of guinea pigs to test for skin sensitization
potential (i.e. TG 406) (13).
DEFINITIONS
3.

Definitions used are provided in Annex 1.

INITIAL CONSIDERATIONS AND LIMITATIONS
4.
The LLNA: BrdU-ELISA is a modified LLNA method for identifying potential skin sensitizing test
substances, with specific limitations. This does not necessarily imply that in all instances the LLNA: BrdUELISA should be used in place of the LLNA or guinea pig tests (i.e. TG 406) (13), but rather that the assay
is of equal merit and may be employed as an alternative in which positive and negative results generally nolonger require further confirmation (10) (11). The testing laboratory should consider all available
information on the test substance prior to conducting the study. Such information will include the identity
and chemical structure of the test substance; its physicochemical properties; the results of any other in vitro
or in vivo toxicity tests on the test substance; and toxicological data on structurally related test substances.
This information should be considered in order to determine whether the LLNA: BrdU-ELISA is© OECD, (2010)
You are free to use this material for personal, non-commercial purposes without seeking prior consent fromthe OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written
permission from the OECD.

442B

OECD/OCDE

appropriate for the test substance (given the incompatibility of limited types of test substances with the
LLNA: BrdU-ELISA [see paragraph 5]) and to aid in dose selection.
5.
The LLNA: BrdU-ELISA is an in vivo method and, as a consequence, will not eliminate the use ofanimals in the assessment of allergic contact sensitizing activity. It has, however, the potential to reduce
the animal use for this purpose when compared to the guinea pig tests (TG 406) (13). Moreover, the
LLNA: BrdU-ELISA offers a substantial refinement of the way in which animals are used for allergic
contact sensitization testing, since unlike the TG 406, the LLNA: BrdU-ELISA does not require thatchallenge-induced dermal hypersensitivity reactions be elicited. Furthermore, the LLNA: BrdU-ELISA
does not require the use of an adjuvant, as is the case for the guinea pig maximisation test (13). Thus, the
LLNA: BrdU-ELISA reduces animal distress. Despite the advantages of the LLNA: BrdU-ELISA over TG
406 (13), there are certain limitations that may necessitate the use of TG 406 (e.g. the testing of certain
metals, false positive findings with certain skin irritants [such as some surfactant-type substances] (6) (1),solubility of the test substance). In addition, test substance classes or substances containing functionalgroups shown to act as potential confounders (15) may necessitate the use of guinea pig tests (i.e. TG 406(13)). Limitations that have been identified for the LLNA (1) have been recommended to apply also to the
LLNA: BrdU-ELISA (10). Other than such identified limitations, the LLNA: BrdU-ELISA should beapplicable for testing any test substances unless there are properties associated with these substances that
may interfere with the accuracy of the LLNA: BrdU-ELISA. In addition, consideration should be given to
the possibility of borderline positive results when Stimulation Index (SI) values between 1.6 and 1.9 are
obtained (see paragraphs 31-32). This is based on the validation database of 43 substances using an
SI ≥ 1.6 (see paragraph 6) for which the LLNA: BrdU-ELISA correctly identified all 32 LLNA sensitizers,
but incorrectly identified two of 11 LLNA non-sensitizers with SI values between 1.6 and 1.9 (i.e.
borderline positive) (10). However, as the same dataset was used for setting the SI-values and calculating
the predictive properties of the test, the stated results may be an over-estimation of the real predictive
properties.
PRINCIPLE OF THE TEST6.
The basic principle underlying the LLNA: BrdU-ELISA is that sensitizers induce proliferation oflymphocytes in the lymph nodes draining the site of test substance application. This proliferation is
proportional to the dose and to the potency of the applied allergen and provides a simple means ofobtaining a quantitative measurement of sensitization. Proliferation is measured by comparing the mean
proliferation in each test group to the mean proliferation in the vehicle treated control group (VC). The
ratio of the mean proliferation in each treated group to that in the concurrent VC group, termed the SI, isdetermined, and should be ≥1.6 before further evaluation of the test substance as a potential skin sensitizer
is warranted. The methods described here are based on the use of measuring BrdU content to indicate anincreased number of proliferating cells in the draining auricular lymph nodes. BrdU is an analogue ofthymidine and is similarly incorporated into the DNA of proliferating cells. The incorporation of BrdU is
measured by ELISA, which utilises an antibody specific for BrdU that is also labelled with peroxidase.
When the substrate is added, the peroxidase reacts with the substrate to produce a coloured product that isquantified at a specific absorbance using a microtiter plate reader.
DESCRIPTION OF THE ASSAY
Selection of animal species
7.
The mouse is the species of choice for this test. Validation studies for the LLNA: BrdU-ELISA
were conducted exclusively with the CBA/JN strain, which is therefore considered the preferred strain (10)(12). Young adult female mice, which are nulliparous and non-pregnant, are used. At the start of the study,
animals should be between 8-12 weeks old, and the weight variation of the animals should be minimal and© OCDE, (2010)2

OECD/OCDE

442B

not exceed 20% of the mean weight. Alternatively, other strains and males may be used when sufficient
data are generated to demonstrate that significant strain and/or gender-specific differences in the LLNA:
BrdU-ELISA response do not exist.
Housing and feeding conditions
8.
Mice should be group-housed (16), unless adequate scientific rationale for housing miceindividually is provided. The temperature of the experimental animal room should be 22 ± 3ºC. Although
the relative humidity should be at least 30% and preferably not exceed 70%, other than during roomcleaning, the aim should be 50-60%. Lighting should be artificial, the sequence being 12 hours light, 12hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking
water.
Preparation of animals
9.
The animals are randomly selected, marked to permit individual identification (but not by any formof ear marking), and kept in their cages for at least five days prior to the start of dosing to allow for
acclimatisation to the laboratory conditions. Prior to the start of treatment all animals are examined toensure that they have no observable skin lesions.
Preparation of dosing solutions
10.
Solid test substances should be dissolved or suspended in solvents/vehicles and diluted, if
appropriate, prior to application to an ear of the mice. Liquid test substances may be applied neat or diluted
prior to dosing. Insoluble substances, such as those generally seen in medical devices, should be subjected
to an exaggerated extraction in an appropriate solvent to reveal all extractable constituents for testing prior
to application to an ear of the mice. Test substances should be prepared daily unless stability data
demonstrate the acceptability of storage.
Reliability check
11.
Positive controls (PC) are used to demonstrate appropriate performance of the assay by responding
with adequate and reproducible sensitivity to a sensitizing test substance for which the magnitude of the
response is well characterised. Inclusion of a concurrent PC is recommended because it demonstrates
competency of the laboratory to successfully conduct each assay and allows for an assessment of intra-,and inter-laboratory reproducibility and comparability. Some regulatory authorities also require a PC foreach study and therefore users are encouraged to consult the relevant authorities prior to conducting the
LLNA: BrdU-ELISA. Accordingly, the routine use of a concurrent PC is encouraged to avoid the need for
additional animal testing to meet such requirements that might arise from the use of a periodic PC (seeparagraph 12). The PC should produce a positive LLNA: BrdU-ELISA response at an exposure level
expected to give an increase in the SI≥ 1.6 over the negative control (NC) group. The PC dose should bechosen such that it does not cause excessive skin irritation or systemic toxicity and the induction isreproducible but not excessive (e.g. SI > 14 would be considered excessive). Preferred PC test substances
are 25% hexyl cinnamic aldehyde (CAS No 101-86-0) and 25% eugenol (CAS No 97-53-0) in
acetone: olive oil (4:1, v/v). There may be circumstances in which, given adequate justification, other PC
test substances, meeting the above criteria, may be used.
12.
While inclusion of a concurrent PC group is recommended, there may be situations in which
periodic testing (i.e. at intervals ≤6 months) of the PC test substance may be adequate for laboratories that
conduct the LLNA: BrdU-ELISA regularly (i.e. conduct the LLNA: BrdU-ELISA at a frequency of no lessthan once per month) and have an established historical PC database that demonstrates the laboratory’s
ability to obtain reproducible and accurate results with PCs. Adequate proficiency with the LLNA: BrdU3© OECD, (2010442B

OECD/OCDEELISA can be successfully demonstrated by generating consistent positive results with the PC in at least 10
independent tests conducted within a reasonable period of time (i.e. less than one year).
13.
A concurrent PC group should always be included when there is a procedural change to the LLNA:
BrdU-ELISA (e.g. change in trained personnel, change in test method materials and/or reagents, change intest method equipment, change in source of test animals), and such changes should be documented inlaboratory reports. Consideration should be given to the impact of these changes on the adequacy of thepreviously established historical database in determining the necessity for establishing a new historical
database to document consistency in the PC results.
14.
Investigators should be aware that the decision to conduct a PC study on a periodic basis instead ofconcurrently has ramifications on the adequacy and acceptability of negative study results generated
without a concurrent PC during the interval between each periodic PC study. For example, if a false
negative result is obtained in the periodic PC study, negative test substance results obtained in the interval
between the last acceptable periodic PC study and the unacceptable periodic PC study may be questioned.
Implications of these outcomes should be carefully considered when determining whether to include
concurrent PCs or to only conduct periodic PCs. Consideration should also be given to using fewer animals
in the concurrent PC group when this is scientifically justified and if the laboratory demonstrates, based onlaboratory-specific historical data, that fewer mice can be used (17).
15.
Although the PC test substance should be tested in the vehicle that is known to elicit a consistent
response (e.g. acetone: olive oil; 4:1, v/v), there may be certain regulatory situations in which testing in a
non-standard vehicle (clinically/chemically relevant formulation) will also be necessary (18). If the
concurrent PC test substance is tested in a different vehicle than the test substance, then a separate VC for
the concurrent PC should be included.
16.
In instances where test substances of a specific chemical class or range of responses are being
evaluated, benchmark test substances may also be useful to demonstrate that the test method is functioning
properly for detecting the skin sensitization potential of these types of test substances. Appropriate
benchmark test substances should have the following properties:•

structural and functional similarity to the class of the test substance being tested;•

known physical/chemical characteristics;•

supporting data from the LLNA: BrdU-ELISA;•

supporting data from other animal models and/or from humans.

TEST PROCEDURE
Number of animals and dose levels
17.
A minimum of four animals is used per dose group, with a minimum of three concentrations of thetest substance, plus a concurrent NC group treated only with the vehicle for the test substance, and a PC
group (concurrent or recent, based on laboratory policy in considering paragraphs 11- 15). Testing multiple
doses of the PC should be considered especially when testing the PC on an intermittent basis. Except forabsence of treatment with the test substance, animals in the control groups should be handled and treated ina manner identical to that of animals in the treatment groups.
18.
Dose and vehicle selection should be based on the recommendations given in the references 2 and19. Consecutive doses are normally selected from an appropriate concentration series such as 100%, 50%,
© OCDE, (2010)

4

OECD/OCDE

442B

25%, 10%, 5%, 2.5%, 1%, 0.5%, etc. Adequate scientific rationale should accompany the selection of theconcentration series used. All existing toxicological information (e.g. acute toxicity and dermal irritation)and structural and physicochemical information on the test substance of interest (and/or structurally related
test substances) should be considered, where available, in selecting the three consecutive concentrations sothat the highest concentration maximises exposure while avoiding systemic toxicity and/or excessive local
skin irritation (19)(20). In the absence of such information, an initial pre-screen test may be necessary (seeparagraphs 21-24).
19.
The vehicle should not interfere with or bias the test result and should be selected on the basis of
maximising the solubility in order to obtain the highest concentration achievable while producing asolution/suspension suitable for application of the test substance. Recommended vehicles are acetone: olive
oil (4:1 v/v), N,N-dimethylformamide, methyl ethyl ketone, propylene glycol, and dimethyl sulphoxide (6)but others may be used if sufficient scientific rationale is provided. In certain situations it may be necessary
to use a clinically relevant solvent or the commercial formulation in which the test substance is marketed
as an additional control. Particular care should be taken to ensure that hydrophilic substances areincorporated into a vehicle system, which wets the skin and does not immediately run off, by incorporationof appropriate solubilisers (e.g. 1% Pluronic® L92). Thus, wholly aqueous vehicles are to be avoided.
20.
The processing of lymph nodes from individual mice allows for the assessment of inter-animal
variability and a statistical comparison of the difference between test substance and VC groupmeasurements (see paragraph 33). In addition, evaluating the possibility of reducing the number of mice in
the PC group is only feasible when individual animal data are collected (17). Further, some national
regulatory authorities require the collection of individual animal data. Regular collection of individual
animal data provides an animal welfare advantage by avoiding duplicate testing that would be necessary if
the test substance results originally collected in one manner (e.g. via pooled animal data) were to beconsidered later by regulatory authorities with other requirements (e.g. individual animal data).
Pre-screen test
21.
In the absence of information to determine the highest dose to be tested (see paragraph 18), a prescreen test should be performed in order to define the appropriate dose level to test in the LLNA: BrdUELISA. The purpose of the pre-screen test is to provide guidance for selecting the maximum dose level touse in the main LLNA: BrdU-ELISA study, where information on the concentration that induces systemic
toxicity (see paragraph 24) and/or excessive local skin irritation (see paragraph 23) is not available. The
maximum dose level tested should be a concentration of 100% of the test substance for liquids or the
maximum possible concentration for solids or suspensions.
22.
The pre-screen test is conducted under conditions identical to the main LLNA: BrdU-ELISA study,
except there is no assessment of lymph node proliferation and fewer animals per dose group can be used.
One or two animals per dose group are suggested. All mice will be observed daily for any clinical signs ofsystemic toxicity or local irritation at the application site. Body weights are recorded pre-test and prior totermination (Day 6). Both ears of each mouse are observed for erythema and scored using Table 1 (20). Ear
thickness measurements are taken using a thickness gauge (e.g. digital micrometer or Peacock Dial
thickness gauge) on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), and Day 6.
Additionally, on Day 6, ear thickness could be determined by ear punch weight determinations, whichshould be performed after the animals are humanely killed. Excessive local irritation is indicated by anerythema score ≥3 and/or ear thickness of ≥25% on any day of measurement (21) (22). The highest dose
selected for the main LLNA: BrdU-ELISA study will be the next lower dose in the pre-screen
concentration series (see paragraph 18) that does not induce systemic toxicity and/or excessive local skinirritation.

5© OECD, (2010442B

OECD/OCDE

Table 1. Erythema Scores
Observation

Score

No erythema

0

Very slight erythema (barely perceptible)

1

Well-defined erythema

2

Moderate to severe erythema

3

Severe erythema (beet redness) to eschar formation preventing grading of erythema

423.
In addition to a 25% increase in ear thickness (21) (22), a statistically significant increase in ear
thickness in the treated mice compared to control mice has also been used to identify irritants in the LLNA(22) (23) (24) (25) (26) (27) (28). However, while statistically significant increases can occur when ear
thickness is less than 25% they have not been associated specifically with excessive irritation (25) (26)(27) (28) (29).
24.
The following clinical observations may indicate systemic toxicity (30) when used as part of an
integrated assessment and therefore may indicate the maximum dose level to use in the main LLNA:
BrdU-ELISA: changes in nervous system function (e.g. pilo-erection, ataxia, tremors, and convulsions);changes in behaviour (e.g. aggressiveness, change in grooming activity, marked change in activity level);changes in respiratory patterns (i.e. changes in frequency and intensity of breathing such as dyspnea,
gasping, and rales), and changes in food and water consumption. In addition, signs of lethargy and/or
unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a >5%
reduction in body weight from Day 1 to Day 6 and mortality should be considered in the evaluation.
Moribund animals or animals showing signs of severe pain and distress should be humanely killed (31).
Main study experimental schedule
25.

The experimental schedule of the assay is as follows:•

Day 1:
Individually identify and record the weight of each animal and any clinical observation.
Apply 25 µL of the appropriate dilution of the test substance, the vehicle alone, or the PC(concurrent or recent, based on laboratory policy in considering paragraphs 11-15), to thedorsum of each ear.

•Days 2 and 3:
Repeat the application procedure carried out on Day 1.

•

Days 4:
No treatment.

•

Days 5:Inject 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution inter-peritoneally.

© OCDE, (2010)

6

OECD/OCDE•

442B

Day 6:
Record the weight of each animal and any clinical observation. Approximately 24 hours(24 h) after BrdU injection, humanely kill the animals. Excise the draining auricular lymph
nodes from each mouse ear and process separately in phosphate buffered saline (PBS) for
each animal. Details and diagrams of the lymph node identification and dissection can befound in reference (17). To further monitor the local skin response in the main study,
additional parameters such as scoring of ear erythema or ear thickness measurements(obtained either by using a thickness gauge, or ear punch weight determinations at
necropsy) may be included into the study protocol.

Preparation of cell suspensions
26.
From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally is
prepared by gentle mechanical disaggregation through 200 micron-mesh stainless steel gauze or anotheracceptable technique for generating a single-cell suspension (e.g. use of a disposable plastic pestle to crush
the lymph nodes followed by passage through a #70 nylon mesh). The procedure for preparing the LNC
suspension is critical in this assay and therefore every operator should establish the skill in advance.
Further, the lymph nodes in NC animals are small, so careful operation is important to avoid any artificial
effects on SI values. In each case, the target volume of the LNC suspension should be adjusted to a
determined optimised volume (approximately 15 mL). The optimised volume is based on achieving a mean
absorbance of the NC group within 0.1- 0.2.
Determination of cellular proliferation (measurement of BrdU content in DNA of lymphocytes)
27.
BrdU is measured by ELISA using a commercial kit (e.g. Roche Applied Science, Mannheim,
Germany, Catalogue Number 11 647 229 001). Briefly, 100 µL of the LNC suspension is added to thewells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU
antibody is added to each well and allowed to react. Subsequently the anti-BrdU antibody is removed bywashing and the substrate solution is then added and allowed to produce chromogen. Absorbance at 370
nm with a reference wavelength of 492 nm is then measured. In all cases, assay test conditions should be
optimised (see paragraph 26).
OBSERVATIONSClinical observations
28.
Each mouse should be carefully observed at least once daily for any clinical signs, either of local
irritation at the application site or of systemic toxicity. All observations are systematically recorded withrecords being maintained for each mouse. Monitoring plans should include criteria to promptly identify
those mice exhibiting systemic toxicity, excessive local skin irritation, or corrosion of skin for euthanasia(31).
Body weights
29.
As stated in paragraph 25, individual animal body weights should be measured at the start of thetest and at the scheduled humane kill.
CALCULATION OF RESULTS

7© OECD, (2010442B

OECD/OCDE

30.
Results for each treatment group are expressed as the mean SI. The SI is derived by dividing themean BrdU labelling index/mouse within each test substance group and the PC group by the mean BrdU
labelling index for the solvent/VC group. The average SI for the VCs is then one.
The BrdU labelling index is defined as:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
Where; em = emission wavelength; and ref = reference wavelength.
31.
The decision process regards a result as positive when SI≥ 1.6 (10). However, the strength of thedose-response relationship, the statistical significance and the consistency of the solvent/vehicle and PC
responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and1.9) is declared positive (3) (6) (32).
32.
For a borderline positive response between an SI of 1.6 and 1.9, users may want to consider
additional information such as dose-response relationship, evidence of systemic toxicity or excessive
irritation, and where appropriate, statistical significance together with SI values to confirm that such results
are positives (10). Consideration should also be given to various properties of the test substance, including
whether it has a structural relationship to known skin sensitizers, whether it causes excessive skin irritationin the mouse, and the nature of the dose-response observed. These and other considerations are discussed
in detail elsewhere (4).
33.
Collecting data at the level of the individual mouse will enable a statistical analysis for presence
and degree of dose-response relationship in the data. Any statistical assessment could include an evaluationof the dose-response relationship as well as suitably adjusted comparisons of test groups (e.g. pair-wise
dosed group versus concurrent solvent/vehicle control comparisons). Statistical analyses may include, e.g.
linear regression or Williams’s test to assess dose-response trends, and Dunnett’s test for pair-wisecomparisons. In choosing an appropriate method of statistical analysis, the investigator should maintain anawareness of possible inequalities of variances and other related problems that may necessitate a datatransformation or a non-parametric statistical analysis. In any case, the investigator may need to carry out
SI calculations and statistical analyses with and without certain data points (sometimes called “outliers”).
DATA AND REPORTING
Data
34.
Data should be summarised in tabular form showing the individual animal BrdU labelling index
values, the group mean BrdU labelling index/animal, its associated error term (e.g. SD, SEM), and themean SI for each dose group compared against the concurrent solvent/vehicle control group.
Test report
35.

The test report should contain the following information:

Test substance and control test substance:–––

identification data (e.g. CAS number, if available; source; purity; known impurities; lot
number);physical nature and physicochemical properties (e.g. volatility, stability, solubility);
if formulation, composition and relative percentages of components;© OCDE, (2010)

8

442B

OECD/OCDE
Solvent/vehicle:––

identification data (purity; concentration, where appropriate; volume used);justification for choice of vehicle;

Test animals:

–
–
–
–

source of CBA mice;
microbiological status of the animals, when known;
number and age of animals;source of animals, housing conditions, diet, etc.;

Test conditions:

–
–
–
–
–
–
–
–
–

source, lot number, and manufacturer’s quality assurance/quality control data (antibody
sensitivity and specificity and the limit of detection) for the ELISA kit;details of test substance preparation and application;
justification for dose selection (including results from pre-screen test, if conducted);
vehicle and test substance concentrations used, and total amount of test substance applied;details of food and water quality (including diet type/source, water source);
details of treatment and sampling schedules;
methods for measurement of toxicity;
criteria for considering studies as positive or negative;
details of any protocol deviations and an explanation on how the deviation affects thestudy design and results;

Reliability check:–––a summary of results of latest reliability check, including information on test substance,
concentration and vehicle used;concurrent and/or historical PC and concurrent negative (solvent/vehicle) control data for
testing laboratory;
if a concurrent PC was not included, the date and laboratory report for the most recent
periodic PC and a report detailing the historical PC data for the laboratory justifying thebasis for not conducting a concurrent PC;

Results:

–
–
–
–
–
–
–

individual weights of mice at start of dosing and at scheduled humane kill; as well as mean
and associated error term (e.g. SD, SEM) for each treatment group;
time course of onset and signs of toxicity, including dermal irritation at site ofadministration, if any, for each animal;a table of individual mouse BrdU labelling indices and SI values for each treatment group;
mean and associated error term (e.g. SD, SEM) for BrdU labelling index/mouse for eachtreatment group and the results of outlier analysis for each treatment group;
calculated SI and an appropriate measure of variability that takes into account the interanimal variability in both the test substance and control groups;
dose-response relationship;
statistical analyses, where appropriate;

Discussion of results:
9© OECD, (2010

442B
–

OECD/OCDEa brief commentary on the results, the dose-response analysis, and statistical analyses,
where appropriate, with a conclusion as to whether the test substance should be considereda skin sensitizer.

© OCDE, (2010)10

OECD/OCDE

442B

LITERATURE(1)OECD (2010), Skin Sensitization: Local Lymph Node Assay, Test Guideline No.
Guidelines for the Testing ofChemicals,OECD,
Paris.
Available[http://www.oecd.org/env/testguidelines](2)

Chamberlain, M. and Basketter, D.A. (1996), The local lymph node assay: status of validation.
Food Chem. Toxicol., 34, 999-1002.

(3)

Basketter, D.A., Gerberick, G.F., Kimber, I. and Loveless, S.E. (1996), The local lymph node
assay: A viable alternative to currently accepted skin sensitisation tests. Food Chem. Toxicol.,34, 985-997.

(4)

Basketter, D.A., Gerberick, G.F. and Kimber, I. (1998), Strategies for identifying false positive
responses in predictive sensitisation tests. Food Chem. Toxicol., 36, 327-33.

(5)

Van Och, F.M.M., Slob, W., De Jong, W.H., Vandebriel, R.J. and Van Loveren, H. (2000), A
quantitative method for assessing the sensitizing potency of low molecular weight chemicals
using a local lymph node assay: employment of a regression method that includes determinationof uncertainty margins. Toxicol., 146, 49-59.

(6)

ICCVAM (1999), The murine local lymph node Assay: A test method for assessing the allergic
contact dermatitis potential of chemicals/compounds: The results of an independent peer review
evaluation coordinated by the Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) and the National Toxicology Program Center for the
Evaluation of Alternative Toxicological Methods (NICETAM). NIH Publication No: 99-4494.
Research Triangle Park, N.C. Available at:[http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf](7)Dean, J.H., Twerdok, L.E., Tice, R.R., Sailstad, D.M., Hattan, D.G., Stokes, W.S. (2001),ICCVAM evaluation of the murine local lymph node assay: II. Conclusions andrecommendations of an independent scientific peer review panel. Reg. Toxicol. Pharmacol.,34(3), 258-273.

(8)

Haneke, K.E., Tice, R.R., Carson, B.L., Margolin, B.H., Stokes, W.S. (2001), ICCVAM
evaluation of the murine local lymph node assay: III. Data analyses completed by the national
toxicology program interagency center for the evaluation of alternative toxicological methods.
Reg. Toxicol. Pharmacol., 34(3), 274-286.

(9)

Sailstad, D.M., Hattan, D., Hill, R.N., Stokes, W.S. (2001), ICCVAM evaluation of the murine
local lymph node assay: I. The ICCVAM review process. Reg. Toxicol. Pharmacol., 34(3), 249257.

(10)

ICCVAM (2010), ICCVAM Test Method Evaluation Report. Nonradioactive local lymph node
assay: BrdU-ELISA Test Method Protocol (LLNA: BrdU-ELISA). NIH Publication No. 107552A/B. Research Triangle Park, NC: National Institute of Environmental Health Sciences.
Available at: [http://iccvam.niehs.nih.gov/methods/immunotox/llna-ELISA/TMER.htm](11)

ICCVAM (2009), Independent Scientific Peer Review Panel Report: Updated validation statusof new versions and applications of the murine local lymph node assay: a test method forassessing the allergic contact dermatitis potential of chemicals and products. Research Triangle
11

429,
at:

© OECD, (2010442B

OECD/OCDE
Park, NC: National Institute of Environmental Health Sciences.
[http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2009.pdf]

Available

at:

(12)

Takeyoshi, M., Iida, K., Shiraishi, K. and Hoshuyama, S. (2005), Novel approach for classifying
chemicals according to skin sensitizing potency by non-radioisotopic modification of the local
lymph node assay. J. Appl. Toxicol., 25, 129-134.

(13)

OECD (1992), Skin Sensitisation, Test Guideline No. 406, Guidelines for Testing of Chemicals,
OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines](14)

Kreiling, R., Hollnagel, H.M., Hareng, L., Eigler, L., Lee, M.S., Griem, P., Dreessen, B., Kleber,
M., Albrecht, A., Garcia, C. and Wendel, A. (2008), Comparison of the skin sensitizing potentialof unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and theguinea pig maximization test (GPMT). Food Chem. Toxicol., 46, 1896-1904.

(15)

Basketter, D., Ball, N., Cagen, S., Carrilo, J.C., Certa, H., Eigler, D., Garcia, C., Esch, H.,
Graham, C., Haux, C., Kreiling, R. and Mehling, A. (2009), Application of a weight of evidence
approach to assessing discordant sensitisation datasets: implications for REACH. Reg.
Toxicol. Pharmacol., 55, 90-96.

(16)

ILAR (1996), Institute of Laboratory Animal Research (ILAR) Guide for the Care and Use ofLaboratory Animals. 7th ed. Washington, DC: National Academies Press.

(17)

ICCVAM (2009), Recommended Performance Standards: Murine Local Lymph Node Assay.
NIH Publication Number 09-7357. Research Triangle Park, NC: National Institute ofEnvironmental Health Sciences. Available at:[http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna-ps/LLNAPerfStds.pdf](18)

McGarry, H.F. (2007), The murine local lymph node assay: regulatory and potency
considerations under REACH. Toxicol., 238, 71-89.

(19)

Kimber, I., Dearman, R.J., Scholes E.W. and Basketter, D.A. (1994), The local lymph node
assay: developments and applications. Toxicol., 93, 13-31.

(20)

OECD (2002), Acute Dermal Irritation/Corrosion, Test Guideline No. 404, Guidelines for
Testing of Chemicals, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines](21)

Reeder, M.K., Broomhead, Y.L., DiDonato, L. and DeGeorge, G.L. (2007), Use of an enhanced
local lymph node assay to correctly classify irritants and false positive substances. Toxicologist,
96, 235.

(22)

ICCVAM (2009), Nonradioactive Murine Local Lymph Node Assay: Flow Cytometry Test
Method Protocol (LLNA: BrdU-FC) Revised Draft Background Review Document. Research
Triangle Park, NC: National Institute of Environmental Health Sciences. Available at:[http://iccvam.niehs.nih.gov/methods/immunotox/fcLLNA/BRDcomplete.pdf].

(23)

Hayes, B.B., Gerber, P.C., Griffey, S.S. and Meade, B.J. (1998), Contact hypersensitivity todicyclohexylcarbodiimide and diisopropylcarbodiimide in female B6C3F1 mice. Drug Chem.
Toxicol., 21, 195-206.

© OCDE, (2010)12

OECD/OCDE

442B

(24)

Homey, B., von Schilling, C., Blumel, J., Schuppe, H.C., Ruzicka, T., Ahr, H.J., Lehmann, P.
and Vohr, V.W. (1998), An integrated model for the differentiation of chemical-induced allergic
and irritant skin reactions. Toxicol. Appl. Pharmacol., 153, 83-94.

(25)

Woolhiser, M.R., Hayes, B.B. and Meade, B.J. (1998), A combined murine local lymph nodeand irritancy assay to predict sensitization and irritancy potential of chemicals. Toxicol. Meth., 8,245-256.

(26)

Hayes, B.B. and Meade, B.J. (1999), Contact sensitivity to selected acrylate compounds in
B6C3F1 mice: relative potency, cross reactivity, and comparison of test methods. Drug. Chem.
Toxicol., 22, 491-506.

(27)

Ehling, G., Hecht, M., Heusener, A., Huesler, J., Gamer, A.O., van Loveren, H., Maurer, T.,
Riecke, K., Ullmann, L., Ulrich, P., Vandebriel, R. and Vohr, H.W. (2005), A European interlaboratory validation of alternative endpoints of the murine local lymph node assay: first round.
Toxicol., 212, 60-68.

(28)Vohr, H.W. and Ahr, H.J. (2005), The local lymph node assay being too sensitive? Arch.
Toxicol., 79, 721-728.

(29)

Patterson, R.M., Noga, E. and Germolec D. (2007), Lack of evidence for contact sensitization by
Pfiesteria extract. Environ. Health Perspect., 115, 1023-1028.

(30)

ICCVAM (2009), Report on the ICCVAM-NICEATM/ECVAM/JaCVAM Scientific Workshop
on Acute Chemical Safety Testing: Advancing In Vitro Approaches and Humane Endpoints for
Systemic Toxicity Evaluations. Research Triangle Park, NC: National Institute of Environmental
Health Sciences. Available at:[http://iccvam.niehs.nih.gov/methods/acutetox/Tox_workshop.htm].

(31)

OECD (2000), Guidance Document on the Recognition, Assessment and Use of Clinical Signs
as Humane Endpoints for Experimental Animals Used in Safety Evaluation, Environmental
Health and Safety Monograph Series on Testing and Assessment No. 19,
ENV/JM/MONO(2000)7, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines](32)

Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Lea, L.,
House, R.V., Ladies, G.S., Loveless, S.E. and Hastings, K.L. (1998), Assessment of the skinsensitization potential of topical medicaments using the local lymph node assay: Aninterlaboratory exercise. J. Toxicol. Environ.l Health, 53, 563-79.

(33)

OECD (2005), Guidance Document on the Validation and International Acceptance of New or
Updated Test Methods for Hazard Assessment, Environment, Health and Safety Monograph
Series on Testing and Assessment No. 34, ENV/JM/MONO(2005)14, OECD, Paris. Available
at: [http://www.oecd.org/env/testguidelines]

13© OECD, (2010

ANNEX 1
Accuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of relevance. The term is often used interchangeably
with “concordance” to mean the proportion of correct outcomes of a test method (33).
Benchmark test substance: A sensitizing or non-sensitizing substance used as a standard for comparison
to a test substance. A benchmark substance should have the following properties: (i) a consistent andreliable source(s); (ii) structural and functional similarity to the class of substances being tested; (iii)
known physical/chemical characteristics; (iv) supporting data on known effects; and (v) known potency inthe range of the desired response.
False negative: A test substance incorrectly identified as negative or non-active by a test method, when infact it is positive or active (33).
False positive: A test substance incorrectly identified as positive or active by a test, when in fact it isnegative or non-active (33).
Hazard: The potential for an adverse health or ecological effect. The adverse effect is manifested only ifthere is an exposure of sufficient level.
Inter-laboratory reproducibility: A measure of the extent to which different qualified laboratories, using
the same protocol and testing the same test substance, can produce qualitatively and quantitatively similar
results. Inter-laboratory reproducibility is determined during the pre-validation and validation processes,
and indicates the extent to which a test can be successfully transferred between laboratories, also referred
to as between-laboratory reproducibility (33).
Intra-laboratory reproducibility: A determination of the extent that qualified people within the samelaboratory can successfully replicate results using a specific protocol at different times. Also referred to aswithin-laboratory reproducibility (33).
Outlier: An outlier is an observation that is markedly different from other values in a random sample froma population.
Quality assurance: A management process by which adherence to laboratory testing standards,
requirements, and record keeping procedures, and the accuracy of data transfer, are assessed by individuals
who are independent from those performing the testing.
Reliability: Measures of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility (33).
Skin sensitization: An immunological process that results when a susceptible individual is exposed
topically to an inducing chemical allergen, which provokes a cutaneous immune response that can lead tothe development of contact sensitization.
Stimulation Index (SI): A value calculated to assess the skin sensitization potential of a test substance thatis the ratio of the proliferation in treated groups to that in the concurrent vehicle control group.

© OCDE, (2010)14

Test substance: Any material tested using this TG, whether it is a single compound or consists of multiple
components (e.g. final products, formulations). When testing formulations, consideration should be given
to the fact that certain regulatory authorities only require testing of the final product formulation. However,there may also be testing requirements for the active ingredient(s) of a product formulation.

15© OECD, (2010ICCVAM Test Method Evaluation Report on the Murine Local
Lymph Node Assay: BrdU-ELISA
A Nonradioactive Alternative Test Method to Assess theAllergic Contact Dermatitis Potential of Chemicals andProducts

Interagency Coordinating Committee on the
Validation of Alternative Methods

National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods

National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

March 2010

NIH Publication Number 10-7552

National Toxicology Program
P.O. Box 12233
Research Triangle Park, NC 27709

ICCVAM LLNA: BrdU-ELISA Evaluation Report

This document is available electronically at:
http://iccvam.niehs.nih.gov/methods/immunotox/llna-ELISA/TMER.htm
When referencing this document, please cite as follows:
ICCVAM. 2010. ICCVAM Test Method Evaluation Report. The Murine Local Lymph Node
Assay: BrdU-ELISA, a Nonradioactive Alternative Test Method to Assess the Allergic Contact
Dermatitis Potential of Chemicals and Products. NIH Publication No. 10-7552. Research Triangle
Park, NC: National Institute of Environmental Health Sciences.

ii

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Table of Contents

0B

List of Tables ......................................................................................................................................... v
List of Figures ....................................................................................................................................... v
List of Abbreviations and Acronyms ................................................................................................ vii
Interagency Coordinating Committee on the Validation of Alternative Methods:
Agency Representatives ............................................................................................................ ix
Acknowledgements ............................................................................................................................... x
Preface ................................................................................................................................................. xv
Executive Summary ......................................................................................................................... xvii
1.0

Introduction ................................................................................................................................ 1

2.0

ICCVAM Recommendations for the Nonradioactive LLNA: BrdU-ELISA Test
Method ........................................................................................................................................ 4

3.0

4.0

5.0

2.1

ICCVAM Recommendations: Test Method Usefulness and Limitations .......................... 4

2.2

ICCVAM Recommendations: Test Method Protocol ........................................................ 4

2.3

ICCVAM Recommendations: Future Studies .................................................................... 5

2.4

ICCVAM Recommendations: Performance Standards ...................................................... 5

Validation Status of the LLNA: BrdU-ELISA Test Method ................................................. 6
3.1

Test Method Description .................................................................................................... 6

3.2

Validation Database ........................................................................................................... 6

3.3

Reference Test Method Data ............................................................................................ 12

3.4

Test Method Accuracy ..................................................................................................... 12

3.5

Test Method Reliability (Intra- and Interlaboratory Reproducibility) ............................. 18

3.6

Animal Welfare Considerations: Reduction, Refinement, and Replacement .................. 19

ICCVAM Consideration of Independent Peer Review Panel Report and Other
Comments ................................................................................................................................. 20
4.1

ICCVAM Consideration of Independent Peer Review Panel Report and OECD
Comments ........................................................................................................................ 20

4.2

ICCVAM Consideration of Public and SACATM Comments ........................................ 22

References ................................................................................................................................. 30

Appendix A

Timeline for ICCVAM Evaluation of the LLNA: BrdU-ELISA ...................... A-1

Appendix B

ICCVAM-Recommended Test Method Protocol: The Murine Local
Lymph Node Assay: 2-Bromodeoxyuridine-ELISA Test Method(LLNA: BrdU-ELISA), a Nonradioactive Alternative Test Method forAssessing the Allergic Contact Dermatitis Potential of Chemicals andProducts................................................................................................................... B-1

iii

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Appendix C

Final Background Review Document: Nonradioactive Murine Local
Lymph Node Assay: BrdU-ELISA Test Method Protocol (LLNA:
BrdU-ELISA) ......................................................................................................... C-1

Appendix D

Independent Scientific Peer Review Panel Assessment ...................................... D-1

Appendix E

Appendix F

iv

D1

Summary Minutes of Independent Scientific Peer Review Panel
Meeting on March 4-6, 2008 ...................................................................... D-3

D2

Peer Review Panel Report: Validation Status of New Versions andApplications of the Murine Local Lymph Node Assay: A Test
Method for Assessing the Allergic Contact Dermatitis Potential ofChemicals and Products (May 2008) ........................................................ D-33

D3

Summary Minutes of Independent Scientific Peer Review Panel
Meeting on April 28-29, 2009 .................................................................. D-73

D4

Independent Scientific Peer Review Panel Report: Updated
Validation Status of New Versions and Applications of the Murine
Local Lymph Node Assay: A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemicals and Products(June 2009) ............................................................................................... D-91

Federal Register Notices and Public Comments ................................................... E-1
E1

Table of Federal Register Notices ............................................................... E-3

E2

Public Comments Received in Response to Federal Register
Notices ....................................................................................................... E-23

E3

SACATM Comments: SACATM Meeting, June 18-19, 2008 ................ E-107

E4

SACATM Comments: SACATM Meeting, June 25-26, 2009 ................ E-121

Relevant Skin Sensitization Regulations and Testing Guidelines....................... F-1
F1

Table of Relevant Skin Sensitization Test Regulations ............................... F-3

F2

EPA Health Effects Test Guidelines OPPTS 870.2600: Skin
Sensitization (March 2003) .......................................................................... F-7

F3

ISO 10993-10: Biological Evaluation of Medical Devices Part 10:
Tests for Irritation and Delayed-type Hypersensitivity (2002) .................. F-25

F4OECD Test Guideline 429: Skin Sensitisation – Local Lymph
Node Assay (Adopted April 2002) ............................................................ F-27

F5OECD Test Guideline 406: Skin Sensitisation (Adopted
July 1992) .................................................................................................. F-37

ICCVAM LLNA: BrdU-ELISA Evaluation Report

List of Tables
Table 3-1

Product Use and Chemical Classification, Traditional LLNA EC3 Values,
LLNA: BrdU-ELISA EC1.6 Values, and Maximum SI Values for 43
Tested Substances ........................................................................................................ 8

Table 3-2

Performance of the LLNA: BrdU-ELISA for 43 Substances in Predicting
Skin Sensitizing Potential Using Alternative Decision Criteria to Identify
Sensitizers................................................................................................................... 13

Table 3-3

Maximum SI Values of 43 Substances Evaluated in the LLNA: BrdUELISA Compared to the Traditional LLNA ............................................................... 16

Table 3-4

Concordance of LLNA: BrdU-ELISA Tests Across Maximum SI
Categories ................................................................................................................... 18

Table 4-1

Opportunities for Public Comment ............................................................................ 22

List of Figures
Figure 3-1

Comparison of LLNA: BrdU-ELISA Stimulation Index with Traditional
LLNA Stimulation Index ........................................................................................... 15v

ICCVAM LLNA: BrdU-ELISA Evaluation Report

This page intentionally left blankvi

ICCVAM LLNA: BrdU-ELISA Evaluation Report

List of Abbreviations and Acronyms
ACD

Allergic contact dermatitis

ACE

AcetoneAOO

Acetone: olive oil (4:1 by volume)

BRD

Background review document

BrdU

Bromodeoxyuridine

CASRN

Chemical Abstracts Service Registry Number

CI

Confidence interval

CMI

5-Chloro-2-methyl-4-isothiazolin-3-one

CPSC

U.S. Consumer Product Safety Commission

CV

Coefficient of variation

DMF

N,N-dimethylformamide

DMSO

Dimethyl sulfoxide

DNCB

Dinitrochlorobenzene

DPCP

Diphenylcyclopropanone

dpm

Disintegrations per minute

EC1.6

Estimated concentration needed to produce a stimulation index of 1.6

EC3

Estimated concentration needed to produce a stimulation index of 3

ECVAM

European Centre for the Validation of Alternative Methods

EGDA

Ethylene glycol dimethacrylate

ELISA

Enzyme-linked immunosorbent assay

EPA

U.S. Environmental Protection Agency

FR

Federal RegisterGP

Guinea pig

GPMTGuinea Pig Maximization Test

3

H

Tritiated

HCA

Hexyl cinnamic aldehyde

ICCVAM

Interagency Coordinating Committee on the Validation of Alternative Methods

ILS

Integrated Laboratory Systems

IWG

Immunotoxicity Working GroupJaCVAM

Japanese Center for the Validation of Alternative Methods

LLNA

Murine local lymph node assay

LLNA:
BrdU-ELISA

Murine local lymph node assay with enzyme-linked immunosorbent assay
detection of bromodeoxyuridine

LNC

Lymph node cells

MAPS

4-methyl aminophenol sulfate

MBT

2-Mercaptobenzothiazolevii

ICCVAM LLNA: BrdU-ELISA Evaluation Report

MEK

Methyl ethyl ketone

NA

Not availableNC

Not calculated

Ni

NickelNICEATM

National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods

NIEHS

National Institute of Environmental Health Sciences

No.

NumberOECD

Organisation for Economic Co-operation and Development

SACATM

Scientific Advisory Committee on Alternative Toxicological Methods

SDStandard deviation

SEM

Standard error of the meanSI

Stimulation index

TG

Test Guideline

U.K.

United Kingdom

U.S.

United States

U.S.C.

United States Codeviii

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Interagency Coordinating Committee on the Validation of
Alternative Methods: Agency Representatives
Agency for Toxic Substances and Disease
Registry* Moiz Mumtaz, Ph.D.
Bruce Fowler, Ph.D.
Edward Murray, Ph.D.
Eric Sampson, Ph.D.
Consumer Product Safety Commission* Marilyn L. Wind, Ph.D. (Chair)+ Kristina Hatlelid, Ph.D.
Joanna Matheson, Ph.D.
Department of Agriculture* Jodie Kulpa‐Eddy, D.V.M. (Vice‐Chair)+ Elizabeth Goldentyer, D.V.M.

Food and Drug Administration
Office of the Commissioner* Suzanne Fitzpatrick, Ph.D., DABTCenter for Biologics Evaluation and Research
Richard McFarland, Ph.D., M.D.
Ying Huang, Ph.D.
Center for Devices and Radiological Health
Melvin E. Stratmeyer, Ph.D.
Vasant G. Malshet, Ph.D., DABTCenter for Drug Evaluation and Research+ Abigail C. Jacobs, Ph.D.
Paul C. Brown, Ph.D.
Center for Food Safety and Applied Nutrition

Department of Defense

David G. Hattan, Ph.D.

* Robert E. Foster, Ph.D.

Robert L. Bronaugh, Ph.D.

+ Patty Decot

Center for Veterinary Medicine

Harry Salem, Ph.D.

Devaraya Jagannath, Ph.D.

Peter J. Schultheiss, D.V.M., DACLAM

M. Cecilia Aguila, D.V.M.

Department of Energy

National Center for Toxicological Research* Michael Kuperberg, Ph.D.

Paul Howard, Ph.D.

+ Marvin Stodolsky, Ph.D.

Donna Mendrick, Ph.D.

Department of the Interior

William T. Allaben, Ph.D. (to Jan. 2009)* Barnett A. Rattner, Ph.D.
+ Sarah Gerould, Ph.D. (to Feb. 2009)

Office of Regulatory Affairs
Lawrence D'Hoostelaere, Ph.D.

ix

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Department of Transportation

National Cancer Institute* George Cushmac, Ph.D.

* T. Kevin Howcroft, Ph.D.

+ Steve Hwang, Ph.D.

Chand Khanna, D.V.M., Ph.D.

Environmental Protection Agency

Alan Poland, M.D. (to Oct. 2008)

Office of Pesticide Programs* John R. “Jack” Fowle III, Ph.D., DABT+ Vicki Dellarco, Ph.D.
+ Tina Levine, Ph.D.
Deborah McCall
Christine Augustyniak, Ph.D. (U.S. Coordinator,
OECD Test Guidelines Program)
Office of Pollution Prevention and Toxics
Jerry Smrchek, Ph.D. (U.S. Coordinator, OECD
Test Guidelines Program, to July 2009)
Office of Research and Development
Suzanne McMaster, Ph.D. (to Dec. 2008)
Julian Preston, Ph.D. (to July 2009)
Stephanie Padilla, Ph.D. (to July 2009)
Office of Science Coordination and Policy

National Institute of Environmental Health
Sciences* William S. Stokes, D.V.M., DACLAM+ Raymond R. Tice, Ph.D.
Rajendra S. Chhabra, Ph.D., DABT
Jerrold J. Heindel, Ph.D.
National Institute for Occupational Safety
and Health* Paul Nicolaysen, V.M.D.
+ K. Murali Rao, M.D., Ph.D.
National Institutes of Health* Margaret D. Snyder, Ph.D.
National Library of Medicine* Pertti (Bert) Hakkinen, Ph.D.
+ Jeanne Goshorn, M.S.

Karen Hamernik, Ph.D. (to July 2009)
Occupational Safety and HealthAdministration* Surender Ahir, Ph.D.
* Principal agency representative+ Alternate principal agency representativex

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Acknowledgements
Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) Immunotoxicity Working Group (IWG)
U.S. Consumer Product Safety Commission
Joanna Matheson, Ph.D. (IWG Co‐Chair)
Marilyn L. Wind, Ph.D.

National Institute of Environmental Health
Sciences
Dori Germolec, Ph.D.
William S. Stokes, D.V.M., DACLAM

U.S. Environmental Protection Agency
Office of Pesticide Programs
Jonathan Chen, Ph.D.

National Institute for Occupational Safety
and Health
B. Jean Meade, D.V.M., Ph.D.

Masih Hashim, D.V.M., Ph.D.
Marianne Lewis
Deborah McCall
Timothy McMahon, Ph.D.
John Redden, M.S.
Jenny Tao, Ph.D.
Office of Pollution Prevention and Toxics
Elizabeth Margosches, Ph.D.
Ronald Ward, Ph.D.
Office of Research and Development

National Library of Medicine
Pertti (Bert) Hakkinen, Ph.D.
European Centre for the Validation of
Alternative Methods – Liaison
Silvia Casati, Ph.D.
Alexandre Angers, Ph.D.
Japanese Center for the Validation of
Alternative Methods – Liaison
Hajime Kojima, Ph.D.

Marsha Ward, Ph.D.
Office of Science Coordination and Policy
Karen Hamernik, Ph.D.
U.S. Food and Drug Administration
Center for Devices and Radiological Health
Vasant G. Malshet, Ph.D., DABTxi

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Jeffrey Toy, Ph.D.
Center for Drug Evaluation and Research
Ruth Barratt, Ph.D., D.V.M.
Paul C. Brown, Ph.D.
Abigail C. Jacobs, Ph.D. (IWG Co‐Chair)
Jiaqin Yao, Ph.D.
Office of Science and Health Coordination
Suzanne Fitzpatrick, Ph.D., DABTxii

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Murine Local Lymph Node Assay
Independent Scientific Peer Review Panel(March 4-6, 2008, and April 28-29, 2009)
Michael Luster, Ph.D. (Panel Chair)
Senior Consultant to the National Institute for
Occupational Safety and Health
Health Effects Laboratory
Morgantown, WVDavid Lovell, Ph.D., FIBiol, CStat, CBiol
Reader in Medical Statistics
Postgraduate Medical School
University of Surrey
Guildford, Surrey, U.K.

Nathalie Alépée, Ph.D.
Scientific Coordinator on Alternatives Methods
in Life Science
L’Oréal Research and Development
Aulnay sous Bois, France

Howard Maibach, M.D.
Professor, Department of Dermatology
University of California – San Francisco
San Francisco, CA

Anne Marie Api, Ph.D.
Vice President, Human Health Sciences
Research Institute for Fragrance Materials
Woodcliff Lake, NJ
Nancy Flournoy, M.S., Ph.D.
Professor and Chair
Department of Mathematics and Statistics
University of Missouri – Columbia
Columbia, MO
Thomas Gebel, Ph.D.1
Regulatory Toxicologist
Federal Institute for Occupational Safety &HealthDortmund, Germany
Kim Headrick, B.Admin., B.Sc.1
International Harmonization and Senior Policy
Advisor
Policy and Programme Service Office
Health Canada
Ottawa, Ontario, Canada
Dagmar Jírová, M.D., Ph.D.
Toxicologist, Research Manager
Head of Reference Center for Cosmetics andReference Laboratory for Experimental
Immunotoxicology
National Institute of Public HealthPrague, Czech RepublicPeter Theran, V.M.D.
Consultant
Massachusetts Society for the Prevention of
Cruelty to Animals
Novato, CA

James McDougal, Ph.D.1
Professor and Director of Toxicology Research
Department of Pharmacology and Toxicology
Boonshoft School of Medicine
Wright State University
Dayton, OH
Michael Olson, Ph.D., A.T.S.
Director of Occupational Toxicology
Corporate Environment, Health and Safety
GlaxoSmithKline
Research Triangle Park, NC
Raymond Pieters, Ph.D.2
Associate Professor
Immunotoxicology Group Leader
Institute for Risk Assessment Sciences
Utrecht University
Utrecht, The Netherlands
Jean Regal, Ph.D.
Professor, Department of Pharmacology
University of Minnesota Medical School
Duluth, MN
Jonathan Richmond, MB ChB, FRCSEd3
Head, Animals Scientific Procedures Division
Home OfficeLondon, U.K.
Michael Woolhiser, Ph.D.
Science and Technology Leader
Toxicology and Environmental Research andConsulting
The Dow Chemical Company
Midland, MI

xiii

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Stephen Ullrich, Ph.D.
Professor of Immunology
Graduate School of Biomedical Sciences
University of Texas
M.D. Anderson Cancer Center – Houston
Houston, TX
1Takahiko Yoshida, M.D., Ph.D.
Professor, Department of Health Science
Asahikawa Medical College
Hokkaido, Japan

Drs. Gebel and McDougal and Ms. Headrick were unable to attend the public meeting on April 28-29,
2009, and did not participate in the review.
2
Dr. Pieters was unable to attend the public meeting on April 28-29, 2009. However, he was involved in
the peer review of the documents and concurred with the conclusions and recommendations included in
the Independent Scientific Peer Review Panel Report – Updated Validation Status of New Versions andApplications of the Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact
Dermatitis Potential of Chemicals and Products.
3
Dr. Richmond was unable to attend the public meeting on March 4-6, 2008. However, he was involved in
the peer review of the documents and concurred with the conclusions and recommendations included in
the Independent Scientific Peer Review Panel Report – Validation Status of New Versions andApplications of the Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact
Dermatitis Potential of Chemicals and Products.

xiv

ICCVAM LLNA: BrdU-ELISA Evaluation Report

National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
William Stokes, D.V.M., DACLAM
Director; Project Officer
Deborah McCarley
Special Assistant; Assistant Project Officer
NICEATM Support Contract Staff (Integrated Laboratory Systems [ILS], Inc.)David Allen, Ph.D.
Thomas Burns, M.S.
Linda Litchfield
Steven Morefield, M.D.
Michael Paris
Eleni Salicru, Ph.D.
Catherine Sprankle
Frank Stack
Judy Strickland, Ph.D., DABT
Linda Wilson
Statistical Consultant for ILS, Inc.
Joseph Haseman, Ph.D.

xv

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Other Acknowledgements
ICCVAM and NICEATM gratefully acknowledge the following individuals and institutions that
submitted data to NICEATM for the evaluation of the LLNA: BrdU-ELISA.
Masahiro Takeyoshi, Ph.D.
Chemicals Evaluation and Research Institute
Saitama, Japanxvi

Hajime Kojima, Ph.D.
Japanese Center for the Validation of
Alternative Methods
Tokyo, JapanICCVAM LLNA: BrdU-ELISA Evaluation Report

Preface
Allergic contact dermatitis (ACD) is an adverse health effect that frequently develops in workers
and consumers exposed to skin sensitizing chemicals and products. ACD results in lost
workdays 1 and can significantly diminish quality of life (Hutchings et al. 2001; Skoet et al.
2003). To minimize the occurrence of ACD, regulatory authorities require testing to identify
substances that may cause skin sensitization. Sensitizing substances must be labeled with adescription of the potential hazard and the precautions necessary to avoid development of ACD.
FF

Skin sensitization testing has typically required the use of guinea pigs (Buehler 1965; Magnusson
and Kligman 1970). However, in 1998, the Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) evaluated and recommended an alternative test
method known as the murine (mouse) local lymph node assay (“traditional LLNA”). 2 The
traditional LLNA provides several advantages compared to guinea pig test methods, including
elimination of potential pain and distress, use of fewer animals, less time to perform, andavailability of dose-response information. Based on the validation database and performance,
ICCVAM recommended the LLNA as an alternative test method for assessing the skinsensitization potential of most types of substances (ICCVAM 1999). United States andinternational regulatory agencies subsequently accepted the traditional LLNA as a valid
alternative test method for ACD testing.
F

F

In 2007, the U.S. Consumer Product Safety Commission (CPSC) requested that ICCVAM
evaluate several modifications of the traditional LLNA, including a nonradioactive version of the
LLNA that measures bromodeoxyuridine (BrdU) incorporation into proliferating lymphocytes byan enzyme-linked immunosorbent assay (ELISA) (referred to hereafter as the “LLNA:
BrdU-ELISA”), instead of using a radioactive marker to measure lymphocyte proliferation. The
BrdU-ELISA was developed by Dr. Masahiro Takayoshi at the Chemicals Evaluation andResearch Institute in Saitama, Japan and validation studies were completed in coordination with
the Japanese Center for the Validation of Alternative Methods (JaCVAM) at the National
Institute of Health Sciences. ICCVAM assigned this activity a high priority after consideringcomments from the public and ICCVAM’s Scientific Advisory Committee on Alternative
Toxicological Methods (SACATM). As part of their ongoing collaboration with ICCVAM,
scientists from the European Centre for the Validation of Alternative Methods (ECVAM) andJaCVAM served as liaisons to the ICCVAM Immunotoxicity Working Group (IWG). A detailed
timeline of the LLNA: BrdU-ELISA evaluation is included with this report.
This Test Method Evaluation Report provides ICCVAM’s recommendations regarding the
LLNA: BrdU-ELISA for assessing the ACD potential of chemicals and products. Since the
LLNA: BrdU-ELISA does not require a radioactive marker, it can be used by laboratories thatcurrently cannot use the traditional LLNA because they do not have a license for using
radioisotopes and in countries that discourage or severely limit the use of radioactive materials.
The report also summarizes the validation status of the LLNA: BrdU-ELISA and provides the
ICCVAM-recommended LLNA: BrdU-ELISA test method protocol.
Following independent scientific peer reviews in 2008 and 2009, ICCVAM submitted a proposed
draft Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG)for the LLNA: BrdU-ELISA that was circulated in July 2009 to the 30 OECD member countries

12

http://www.blf.gov/IIF
The “traditional LLNA” refers to the validated ICCVAM-recommended LLNA test method protocol,
which measures lymphocyte proliferation based on incorporation of 3H-methyl thymidine or 125Iiododeoxyuridine into the cells of the draining auricular lymph nodes (ICCVAM 1999; Dean et al. 2001).
xvii

ICCVAM LLNA: BrdU-ELISA Evaluation Reportfor review and comment. The U.S. CPSC and NICEATM-ICCVAM hosted an OECD Expert
Consultation meeting on October 20-22, 2009, to evaluate the comments. A revised TG was
distributed to the 30 OECD member countries in December 2009 for comment and then the final
draft was forwarded to the OECD Working Group of National Co-ordinators of the Test
Guidelines Programme, which was approved as TG 442B at their March 23-25, 2010 meeting.
ICCVAM solicited and considered public comments and stakeholder involvement throughout the
LLNA: BrdU-ELISA evaluation process. ICCVAM considered the SACATM comments, theconclusions of the Panel and the OECD Expert Consultation, and all public comments before
finalizing the ICCVAM test method recommendations for the LLNA: BrdU-ELISA. The
recommendations and the background review document (BRD), which is provided as an appendix
to this report, are incorporated in this ICCVAM Test Method Evaluation Report. As required by
the ICCVAM Authorization Act, ICCVAM will forward its recommendations to U.S. Federal
agencies for consideration. Federal agencies must respond to ICCVAM within 180 days after
receiving the ICCVAM test method recommendations. ICCVAM recommendations are available
to the public on the NICEATM-ICCVAM website, 3 and agency responses will also be madeavailable on the website as they are received.
FF

We gratefully acknowledge the many individuals who contributed to the preparation, review, andrevision of this report. We especially recognize the Panel members for their thoughtful
evaluations and generous contributions of time and effort. Special thanks are extended to
Dr. Michael Luster for serving as the Panel Chair and to Dr. Michael Woolhiser, Dr. Michael
Olson, Dr. Stephen Ullrich, and Kim Headrick for their service as Evaluation Group Chairs. We
thank the IWG for assuring a meaningful and comprehensive review. We especially thank Dr.
Joanna Matheson (Consumer Product Safety Commission) and Dr. Abigail Jacobs (U.S. Food andDrug Administration Center for Drug Evaluation and Research) for serving as Co-Chairs of the
IWG. We also acknowledge Integrated Laboratory Systems, Inc., the NICEATM support
contractor, for providing excellent scientific and operational support, including Dr. David Allen,
Thomas Burns, Michael Paris, Dr. Eleni Salicru, Frank Stack, and Dr. Judy Strickland. Finally,
we thank Dr. Silvia Casati and Dr. Hajime Kojima, the IWG liaisons from ECVAM and
JaCVAM, respectively, for their participation and contributions.
This comprehensive ICCVAM evaluation of the LLNA: BrdU-ELISA should facilitate regulatory
agency decisions on the acceptability of the method. Use of the method by industry can be
expected to significantly reduce and refine animal use for ACD testing while continuing tosupport the protection of human health.
Marilyn Wind, Ph.D.
Deputy Associate Executive Director
Directorate for Health Sciences
U.S. Consumer Product Safety Commission
Chair, ICCVAM
William S. Stokes, D.V.M., DACLAM
Rear Admiral/Assistant Surgeon General, U.S. Public Health Service
Director, NICEATM
Executive Director, ICCVAM

3

Available at http://iccvam.niehs.nih.gov/methods/immunotox/llna-ELISA/TMER.htm

xviii

ICCVAM LLNA: BrdU-ELISA Evaluation Report

Executive Summary
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
recently evaluated the validation status of a nonradioactive version of the murine local lymph
node assay (LLNA) called the LLNA: BrdU-ELISA. The LLNA is used to identify chemicals andproducts that may cause allergic contact dermatitis (ACD), an allergic skin reaction characterized
by redness, swelling, and itching. The LLNA: BrdU-ELISA uses bromodeoxyuridine (BrdU)
uptake to measure proliferating lymphocytes. The BrdU in this version is quantified with an
enzyme-linked immunosorbent assay (ELISA) kit, while the traditional LLNA uses 3H-methyl
thymidine or 125I-iododeoxyuridine uptake to measure lymphocyte proliferation. 4 This Test
Method Evaluation Report provides ICCVAM’s recommendations regarding the usefulness andlimitations of the LLNA: BrdU-ELISA as an alternative to the traditional LLNA. The report
includes the ICCVAM-recommended LLNA: BrdU-ELISA test method protocol, the final
LLNA: BrdU-ELISA background review document (BRD) describing the validation status of thetest method, and recommendations for future studies and performance standards.
F

F

Following nomination of the LLNA: BrdU-ELISA by the U.S. Consumer Product Safety
Commission (CPSC), the National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM), ICCVAM, and the ICCVAM Immunotoxicity
Working Group prepared an initial draft BRD and draft test method recommendations. The drafts
were provided to an independent international scientific peer review panel (Panel) and to the
public for comment. The Panel met twice in public session to review the initial and revised draft
BRD and draft ICCVAM recommendations. The initial draft BRD evaluated data for 24
substances. The Panel initially met in public session on March 4-6, 2008, to discuss its peer
review of the ICCVAM draft BRD and to provide conclusions and recommendations regarding
the validation status of the LLNA: BrdU-ELISA test method. The Panel also reviewed how well
the information in the draft BRD supported ICCVAM’s draft test method recommendations. The
Panel concluded that definitive test method recommendations could not be made until a detailed
protocol and individual animal data were obtained and an evaluation of interlaboratory
reproducibility was conducted.
NICEATM revised the draft BRD with additional information and data. The revised draft BRD
evaluated data for 31 substances. The Panel reconvened in public session on April 28-29, 2009, toreview the ICCVAM revised draft BRD and to finalize its conclusions and recommendations on
the current validation status of the LLNA: BrdU-ELISA test method.
Based on the revised draft ICCVAM recommendations and Panel reports, NICEATM submitted a
proposed draft Organisation for Economic Co-operation and Development (OECD) Test
Guideline (TG) for the LLNA: BrdU-ELISA. The draft TG was circulated in July 2009 to the 30
OECD member countries for review and comment. The U.S. CPSC and NICEATM-ICCVAM
hosted an OECD Expert Consultation meeting on October 20-22, 2009, to evaluate the comments.
The expert group reviewed the draft OECD TG for the LLNA: BrdU-ELISA, proposed responses
to comments from member countries, and evaluated LLNA: BrdU-ELISA results for 12
additional substances tested and submitted to NICEATM after the April 2009 Panel evaluation. A
revised TG was distributed to the 30 OECD member countries in December 2009 for commentand then the final draft was forwarded to the OECD Working Group of National Co-ordinators of

4

The traditional LLNA refers to the validated ICCVAM-recommended LLNA protocol, which measures
lymphocyte proliferation based on incorporation of 3H methyl thymidine or 125I-iododeoxyuridine into
the cells of the draining auricular lymph nodes (ICCVAM 1999; Dean et al. 2001).
xix

ICCVAM LLNA: BrdU-ELISA Evaluation Report

the Test Guidelines Programme, which approved the LLNA: BrdU ELISA as TG442B at their
March 23-25, 2010 meeting.
In finalizing this Test Method Evaluation Report and the BRD, which is included as an appendix,
ICCVAM considered (1) the conclusions and recommendations of the Panel and the OECD
Expert Consultation, (2) comments from ICCVAM’s Scientific Advisory Committee on
Alternative Toxicological Methods (SACATM), and (3) public comments.
ICCVAM Recommendations: Test Method Usefulness and Limitations
ICCVAM concludes that the accuracy and reliability of the LLNA: BrdU-ELISA support use of
the test method to identify substances as potential skin sensitizers or nonsensitizers. For thevalidation database of 43 substances, the LLNA: BrdU-ELISA correctly identified all 32 LLNA
sensitizers (0% [0/32] false negatives), and nine of the 11 LLNA nonsensitizers (18% [2/11] false
positives). ICCVAM recommends that a stimulation index (SI) ≥ 1.6 be used as the decision
criterion to identify substances as potential sensitizers. ICCVAM bases this recommendation on
the fact that no false negatives, relative to the traditional LLNA, result with the current validation
database when SI ≥ 1.6 is used.
A limitation of the LLNA: BrdU-ELISA is the potential for false positive results when borderline
positive responses between an SI of 1.6 and 1.9 are obtained (see Section 3.4). ICCVAM
considers the applicability domain for the LLNA: BrdU-ELISA to be the same as the traditional
LLNA unless there are properties associated with a class of materials that may interfere with theaccuracy of the LLNA: BrdU-ELISA. One exception would be nickel compounds. Unlike thetraditional LLNA, the LLNA: BrdU-ELISA can be used for testing nickel compounds based on
its ability to correctly identify them as potential sensitizers.
ICCVAM Recommendations: Test Method Protocol
ICCVAM recommends a LLNA: BrdU-ELISA test method protocol that is based on the protocol
developed by Takeyoshi et al. (2001) and refined during an interlaboratory validation study(Kojima et al. 2008). The ICCVAM-recommended LLNA: BrdU-ELISA protocol incorporates all
aspects of the ICCVAM-recommended traditional LLNA test method protocol, except for those
procedures unique to the conduct of the LLNA: BrdU-ELISA. In testing situations where doseresponse information is not required, or negative results are anticipated, ICCVAM recommends
that the reduced LLNA: BrdU-ELISA should be considered and used where determined
appropriate. The reduced LLNA tests only the high dose, thus further reducing animal use by up to40%.
ICCVAM Recommendations: Future Studies
ICCVAM recommends the following future studies to further characterize the usefulness andlimitations of the LLNA: BrdU-ELISA test method:
•

•
•

xx

Efforts should be made to identify additional human data and human experience fortest substances. These data may be used to further assess the usefulness andlimitations of this and other versions of the LLNA for identifying human sensitizing
substances. Such efforts might include post-marketing surveillance of consumers forallergic reactions and occupational surveillance of potentially exposed workers.
Additional substances that are nonsensitizing skin irritants should be tested todetermine the impact of such substances on the false positive rate of the LLNA:
BrdU-ELISA.
Efforts should be made to further characterize the sensitization potential of borderline
positive substances (those that produce an SI between 1.6 and 1.9) in the LLNA:
BrdU-ELISA to determine if such results might be false positives. This could include
evaluations of peptide reactivity, determination of molecular weight, identification of

ICCVAM LLNA: BrdU-ELISA Evaluation Reportresults from related chemicals, human studies where ethically and scientificallyjustified, review of occupational exposures and postmarketing experience ormonitoring, or in vitro testing data. All decision criteria should be reassessed asadditional discriminators and data become available.
ICCVAM Recommendations: Performance Standards
ICCVAM concludes that the ICCVAM-recommended performance standards (ICCVAM 2009a)
for the traditional LLNA can be used to evaluate any future modifications of the LLNA: BrdUELISA. The ICCVAM-recommended performance standards for the traditional LLNA apply to
the LLNA: BrdU-ELISA because the test method is functionally and mechanistically similar to
the traditional LLNA.
Validation Status of the LLNA: BrdU-ELISA
The mechanistic basis of the LLNA: BrdU-ELISA is identical to that of the traditional LLNA.
The traditional LLNA measures the lymphocyte proliferation in the draining lymph nodes for theskin area where the test article is applied. In the traditional LLNA, lymphocyte proliferation morethan three-fold or higher than the vehicle control is considered a positive response indicative of askin sensitizing substance. The only difference between the test method protocols for the
traditional LLNA and the LLNA: BrdU-ELISA is the procedure for measuring lymphocyte
proliferation. The traditional LLNA assesses lymphocyte proliferation by measuring theincorporation of radioactivity into the DNA of dividing cells in the draining auricular lymph
nodes. The LLNA: BrdU-ELISA assesses cell proliferation by measuring the incorporation of anonradioactive thymidine analog, BrdU, into the DNA of dividing cells using an ELISA.
The accuracy of the LLNA: BrdU-ELISA was compared to that of the traditional LLNA using thecurrent validation database of 43 test substances. Optimal LLNA: BrdU-ELISA performance wasachieved using SI ≥ 1.6 to classify sensitizers versus nonsensitizers. Compared to the traditional
LLNA, accuracy was 95% (41/43), with a false positive rate of 18% (2/11) and a false negative
rate of 0% (0/32). The two false positive substances produced SI values between 1.6 and 1.9 in
the LLNA: BrdU-ELISA. Therefore, other available information such as dose-response, evidenceof systemic toxicity or excessive local irritation, and where appropriate, statistical significancetogether with SI values should be considered to confirm that such borderline positive results arepotential skin sensitizers. Consideration should also be given to various properties of the test
substance, including whether it is structurally similar to known skin sensitizers.
An evaluation to determine the robustness of the SI ≥ 1.6 decision criterion indicated that the SI
was quite stable. Taking different samples of the data as training and validation sets had relatively
little impact on the cutoff SI criteria or on the resulting number of false positives or false
negatives.
ICCVAM concludes that the reproducibility of the LLNA: BrdU-ELISA supports the use of themethod to identify substances as potential skin sensitizers and nonsensitizers. The validation
database supported an assessment of both intra-and interlaboratory reproducibility. One study was
conducted to assess interlaboratory reproducibility.
In a qualitative analysis of intralaboratory reproducibility, two to six LLNA: BrdU-ELISA tests
yielded 100% concordance for sensitizer/nonsensitizer outcomes for 10/12 substances (10sensitizers and two nonsensitizers). One of the nonsensitizers with 100% concordance, however,produced false positive results in 2/2 tests. The two discordant substances were traditional LLNA
sensitizers that yielded one test with SI < 1.6 and another test with SI > 1.6. Quantitative analyses
of EC1.6 values (estimated concentration needed to produce an SI of 1.6) were performed for
four substances tested two to five times. The analyses produced coefficient of variation (CV)values from 37% to 118%.

xxi

ICCVAM LLNA: BrdU-ELISA Evaluation Report

The qualitative interlaboratory reproducibility analysis of 10 substances (seven sensitizers andthree nonsensitizers) tested in three to seven laboratories indicated 100% interlaboratory
agreement (3/3, 6/6, or 7/7) for nine substances (seven sensitizers and two nonsensitizers). One of
the nonsensitizers with 100% concordance, however, produced false positive results in 3/3
laboratories. There was 67% (4/6) agreement among the tests for the remaining nonsensitizer.
Interlaboratory CV values for the EC1.6 values of the seven sensitizers ranged from 31% to 93%.
Reproducibility of results for the 18 substances (13 LLNA sensitizers and 5 LLNA
nonsensitizers) that had two to 12 test results, regardless of whether the tests were performed in
one laboratory or multiple laboratories, was assessed with respect to SI category. When the SI ≥1.6 decision criterion was used to classify sensitizers and nonsensitizers, the results for 78%(14/18) of the substances were 100% concordant. The results for 85% (11/13) of the LLNA
sensitizers were 100% concordant (i.e., all yielded SI ≥ 1.6) for two to 12 tests. The results for
60% (3/5) of the nonsensitizers were 100% concordant for two to three tests. All (3/3) tests for
two nonsensitizers had SI < 1.6. All (2/2) tests for the third nonsensitizer yielded SI values
between 1.6 and 1.9, the narrow region in which false positive results occurred.
The Panel agreed with ICCVAM that the reproducibility of the LLNA: BrdU-ELISA supported
the use of the method to identify substances as potential skin sensitizers and nonsensitizers.
ICCVAM Consideration of Independent Peer Review Panel Report and Other Comments
The ICCVAM evaluation process incorporates a high level of scientific peer review andtransparency. The evaluation process for the LLNA: BrdU-ELISA included two public review
meetings by an independent scientific peer review panel, multiple opportunities for publiccomments, consideration of the OECD Expert Consultation on the LLNA, and comments fromthe SACATM. ICCVAM and the Immunotoxicity Working Group considered the Panel report,
conclusions of the OECD Expert Consultation, the SACATM comments, and all publiccomments before finalizing the ICCVAM Test Method Evaluation Report and final BRD for the
LLNA: BrdU-ELISA.

xxii

1.0

Introduction

0B

The murine local lymph node assay (traditional LLNA 1 ) is an alternative skin sensitization test
method that requires fewer animals and less time than currently accepted guinea pig (GP) tests (e.g.,
the guinea pig maximization test [GPMT] and the Buehler test). It also avoids animal discomfort thatcan occur in the guinea pig tests when substances cause allergic contact dermatitis (ACD). The LLNA
measures cell proliferation in the draining auricular lymph nodes of the mouse by analyzing
incorporation of a radioactive marker into newly synthesized DNA. The LLNA was the firstalternative test method evaluated and recommended by the U.S. Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM). International regulatory authorities have now
recognized the traditional LLNA as an acceptable alternative to GP tests for most testing situations.
F

F

The LLNA with detection of bromodeoxyuridine (BrdU) incorporation by an enzyme-linked
immunosorbent assay (ELISA) (referred to hereafter as the “LLNA: BrdU-ELISA”) was one ofseveral modified versions of the LLNA nominated by the U.S. Consumer Product Safety Commission(CPSC) for evaluation by ICCVAM and the National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM). 2 It is a nonradioactive version of the
LLNA that assesses cell proliferation using the incorporation of BrdU into newly synthesized DNA
rather than by quantifying the incorporation of 3H-methyl thymidine or 125I-iododeoxyuridine. The
increase in BrdU in lymph nodes from test animals compared to vehicle controls is then quantified
using an ELISA kit. The LLNA: BrdU-ELISA can reduce the use of animals for skin sensitization
testing when it is used in place of GP tests in countries that severely limit or discourage the use ofradioactive materials that are required by the traditional LLNA.
F

F

In accordance with the ICCVAM Authorization Act of 2000 (Public Law 106-545, 42 United States
Code 285l-3), ICCVAM coordinates the technical evaluation of new, revised, and alternative test
methods with regulatory applicability. After considering comments from the public and ICCVAM’s
advisory committee, the Scientific Advisory Committee on Alternative Toxicological Methods(SACATM), ICCVAM members unanimously agreed that the LLNA: BrdU-ELISA should have ahigh priority for evaluation. A detailed timeline of the LLNA: BrdU-ELISA evaluation is provided inAppendix A. The ICCVAM-recommended LLNA: BrdU-ELISA test method protocol and the final
LLNA: BrdU-ELISA background review document (BRD) are provided in Appendices B and C,
respectively.
The ICCVAM Immunotoxicity Working Group (IWG) was established to work with NICEATM toevaluate the LLNA: BrdU-ELISA and other test methods and applications. The European Centre for
the Validation of Alternative Methods (ECVAM) and the Japanese Center for the Validation of
Alternative Methods (JaCVAM) designated liaison members for the IWG.
To facilitate peer review of the LLNA: BrdU-ELISA test method, the IWG and NICEATM prepared
a comprehensive draft BRD that provided information and data from validation studies and thescientific literature. A May 17, 2007, Federal Register (FR) notice (72 FR 27815 3 ) requested data andinformation on these test methods and nominations of individuals to serve on an international
independent scientific peer review panel (Panel). The request was also disseminated via the ICCVAM
electronic mailing list and through direct requests to over 100 stakeholders. In response to thisrequest, one individual submitted LLNA: BrdU-ELISA data and three individuals or organizations
nominated members to the Panel (see Section 4.0).
F

1

F

The “traditional LLNA” refers to the validated ICCVAM-recommended LLNA test method protocol, whichmeasures lymphocyte proliferation based on incorporation of 3H-methyl thymidine or 125I-iododeoxyuridine
into the cells of the draining auricular lymph nodes (ICCVAM 1999; Dean et al. 2001).
2
Available at http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
3
Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf

In the initial draft BRD, ICCVAM examined data for 24 substances (16 sensitizers and eight
nonsensitizers, as classified by the traditional LLNA) that were tested in a single laboratory, withresults reported among six published studies and one platform presentation. On January 8, 2008,
ICCVAM announced the availability of the draft BRD to the public and a public Panel meeting toreview the validation status of the LLNA: BrdU-ELISA (and other LLNA-related activities)(73 FR 1360 4 ). All of the information provided to the Panel, including the ICCVAM draft BRD, draft
test method recommendations, and all public comments received prior to the Panel meeting, weremade publicly available via the NICEATM–ICCVAM website. 5
FFF

The first Panel meeting was a public session held on March 4–6, 2008, to review the validation statusof the LLNA: BrdU-ELISA and the completeness of the ICCVAM draft BRD (see Appendix D1).
The Panel evaluated (1) the extent to which the draft BRD addressed established validation andacceptance criteria and (2) the extent to which the BRD supported ICCVAM’s draft proposed test
method uses, recommended test method protocol, draft test method performance standards, andproposed future studies. Interested stakeholders from the public were provided opportunities tocomment at the Panel meeting. The Panel considered these comments as well as those submitted prior
to the meeting before concluding their deliberations. The Panel agreed with the draft ICCVAM
recommendations that the LLNA: BrdU-ELISA may be useful for identifying substances as potential
skin sensitizers and nonsensitizers, but that more information and data were needed before definitive
conclusions on the usefulness and limitations of the LLNA: BrdU-ELISA could be made. The Panelnoted that the following information was needed before definitive recommendations could be made:1) a detailed test method protocol; 2) individual animal data on a larger set of balanced reference
substances with respect to physicochemical properties and sensitization potency; and 3) an evaluationof interlaboratory reproducibility. On May 20, 2008, ICCVAM posted a report of the Panel’s
recommendations 6 (see Appendix D2) on the NICEATM–ICCVAM website for public review andcomment (announced in 73 FR 29136 7 ).
F

F

F

F

ICCVAM provided SACATM with the draft BRD and draft test method recommendations, the Panelreport, and all public comments for discussion at their meeting on June 18–19, 2008, where public
stakeholders were given another opportunity to comment.
NICEATM subsequently obtained a detailed test method protocol and additional data and revised thedraft BRD to include this new information. The revised draft BRD included an accuracy evaluation
for the expanded database of individual animal results for 31 substances (22 sensitizers and nine
nonsensitizers, as classified by the traditional LLNA) as well as an evaluation of interlaboratory
reproducibility. Based on the analyses included in the revised draft BRD, ICCVAM prepared revised
draft test method recommendations for proposed test method uses and limitations, recommended test
method protocol, test method performance standards, and future studies for the LLNA: BrdU-ELISA.
ICCVAM released the revised draft documents to the public for comment on February 27, 2009, andannounced a second meeting of the Panel (74 FR 8974 8 ). The Panel reconvened on April 27-28,
2009, to reassess the validation status of the LLNA: BrdU-ELISA (see Appendix D3). The Panel also
reviewed the completeness of the revised draft ICCVAM BRD and the extent to which theinformation therein supported the revised draft ICCVAM test method recommendations. On
June 1, 2009, ICCVAM posted the second report of the Panel’s recommendations 9 (see
F

F

F

4

F

Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_25553.pdf
http://iccvam.niehs.nih.gov
6
Available at http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf
7Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR-E8-11195.pdf
8Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR-E9-4280.pdf
9Available at http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2009.pdf
5Appendix D4) on the NICEATM-ICCVAM website for public review and comment (announced in
74 FR 26242 10 ).
F

F

ICCVAM provided SACATM with the revised draft BRD, the second Panel report, and all publiccomments for discussion at their meeting on June 25-26, 2009, where public stakeholders were given
another opportunity to comment.
Based on the revised draft ICCVAM recommendations, NICEATM submitted a proposed draft
Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) for the
LLNA: BrdU-ELISA that was circulated in July 2009 to the 30 OECD member countries for review
and comment via their National Co-ordinators, who distributed the draft TG to interested
stakeholders. An OECD Expert Consultation meeting was held on October 20-22, 2009, to evaluate
the comments. Scientists from the National Institute of Environmental Health Sciences, theEnvironmental Protection Agency, the Food and Drug Administration, and CPSC, as well as U.S. andinternational experts from industry and other stakeholder organizations, participated in the meeting,which was co-hosted by CPSC and NICEATM-ICCVAM. The expert group reviewed the draft
OECD TG for the LLNA: BrdU-ELISA, proposed responses to comments from member countries,and evaluated additional LLNA: BrdU-ELISA results for substances tested and submitted to
NICEATM after the Panel evaluation. The expert group convened a subsequent teleconference on
December 1, 2009, to discuss outstanding issues identified at the October meeting. A revised TG wasagain distributed to the 30 OECD member countries in December 2009 for review and comment by
national experts and interested stakeholders. A final teleconference of the Expert Consultation wasconvened on January 29, 2010, to discuss the member country comments received during the lastround of review, and a final draft TG was developed based on these discussions. This final draft was
forwarded to the OECD Working Group of National Co-ordinators of the Test Guidelines Programme
to consider for adoption at their March 23-25, 2010, meeting.
ICCVAM and the IWG considered the SACATM comments, the Panel report, conclusions of theOECD Expert Consultation, and all public comments before finalizing ICCVAM test method
recommendations for the LLNA: BrdU-ELISA. The recommendations (Section 2) and the final BRD(Appendix C) are incorporated in this ICCVAM Test Method Evaluation Report. As required by the
ICCVAM Authorization Act (2000; Public Law 106-545, 42 United States Code 285l-3), ICCVAM
will forward its recommendations to U.S. Federal agencies for consideration. Federal agencies must
respond to ICCVAM within 180 days after receiving ICCVAM test method recommendations.
ICCVAM recommendations are available to the public on the NICEATM-ICCVAM website, andagency responses also will be made available on the website as they are received.

10

Announced in 74 FR 26242 http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR-E9-12360.pdf

2.0

ICCVAM Recommendations for the Nonradioactive LLNA: BrdUELISA Test Method
1B

ICCVAM evaluated the validation status of the LLNA: BrdU-ELISA as a nonradioactive
modification of the traditional LLNA (ICCVAM 1999; Sailstad et al. 2001; Dean et al. 2001 Haneke
et al. 2001) to identify substances that may cause ACD for regulatory hazard classification andlabeling purposes. While the traditional LLNA assesses cellular proliferation by measuring theincorporation of 3H-methyl thymidine or 125I-iododeoxyuridine into the DNA of dividing lymph nodecells, the LLNA: BrdU-ELISA assesses cellular proliferation by measuring the incorporation of thethymidine analog BrdU using ELISA detection (see Appendix B). NICEATM and ICCVAM
prepared a comprehensive report on the data and information supporting the validity of this test
method, including its accuracy and reliability compared to the traditional LLNA (see Section 3.0 andAppendix C).

2.1

ICCVAM Recommendations: Test Method Usefulness and Limitations

5B

ICCVAM concludes that the accuracy and reliability of the LLNA: BrdU-ELISA supports the use of
the test method to identify substances as potential skin sensitizers and nonsensitizers. For thevalidation database of 43 substances, 11 the LLNA: BrdU-ELISA correctly identified all 32 LLNA
sensitizers (0% [0/32] false negatives), and nine of the 11 LLNA nonsensitizers (18% [2/11] false
positives). ICCVAM recommends that a stimulation index (SI) ≥ 1.6 be used as the decision criterion
to identify substances as potential sensitizers. ICCVAM bases this recommendation on the fact thatno false negatives, relative to the traditional LLNA, result with the current validation database whenan SI ≥ 1.6 is used.
FF

A limitation of the LLNA: BrdU-ELISA is the potential for false positive results when borderline
positive responses between an SI of 1.6 and 1.9 are obtained (see Section 3.4). ICCVAM considers
the applicability domain for the LLNA: BrdU-ELISA to be the same as the traditional LLNA unlessthere are properties associated with a class of materials that may interfere with the accuracy of the
LLNA: BrdU-ELISA. One exception would be nickel compounds where, unlike the traditional
LLNA, the LLNA: BrdU-ELISA can be used for testing nickel compounds based on its ability tocorrectly identify them as potential sensitizers.

2.2

ICCVAM Recommendations: Test Method Protocol

6B

ICCVAM recommends a LLNA: BrdU-ELISA test method protocol (Appendix B) that was based on
the protocol developed by Takeyoshi et al. (2001) and refined during an interlaboratory validation
study (Kojima et al. 2008). The ICCVAM-recommended LLNA: BrdU-ELISA protocol incorporates
all aspects of the ICCVAM-recommended LLNA test method protocol (Appendix A of ICCVAM
2009a), except for those procedures unique to the conduct of the LLNA: BrdU-ELISA. Key aspects
included in the ICCVAM-recommended protocol include the following:
•

•
•
11

The high dose should be the maximum possible concentration (for liquids, solids, or
suspensions) that does not produce systemic toxicity and/or excessive local skin irritation.
The measurement of ear thickness is a potentially valuable adjunct for identifying local
skin irritation.
A minimum of four animals per dose group is recommended.
Collection of individual animal data is recommended.

For the accuracy analyses, results for substances tested multiple times were combined so that each substance
was represented by one result. In this case, the single result used for each substance represented the mostprevalent outcome. Multiple tests were available for 18 substances tested with the LLNA: BrdU-ELISA.

•

Inclusion of a concurrent vehicle control and concurrent positive control in each study isrecommended.

Additionally, ICCVAM recommends there should be a measure of variability of the positive control
response over time. Laboratories should maintain a historical database of positive control SI values
such that results can be compared to the mean historical SI. There could be cause for concern when anegative test substance result is accompanied by a concurrent positive control SI value significantly
lower than the mean historical SI.
In testing situations where dose-response information is not required, or negative results are
anticipated, ICCVAM recommends that the reduced LLNA: BrdU-ELISA should be considered andused where determined appropriate. The reduced LLNA: BrdU-ELISA protocol uses only the highdose (Kimber et al. 2006; ESAC 2007; ICCVAM 2009b), thus further reducing animal use by up to
40%.

2.3

ICCVAM Recommendations: Future Studies

7B

ICCVAM recommends the following future studies to further characterize the usefulness andlimitations of the LLNA: BrdU-ELISA test method:
•

•
•

2.4

Efforts should be made to identify additional human data and human experience for test
substances. These data may be used to further assess the usefulness and limitations of thisand other versions of the LLNA for identifying human sensitizing substances. Suchefforts might include post-marketing surveillance of consumers for allergic reactions andoccupational surveillance of potentially exposed workers.
Additional substances that are nonsensitizing skin irritants should be tested to determine
the impact of such substances on the false positive rate of the LLNA: BrdU-ELISA.
Efforts should be made to further characterize the sensitization potential of borderline
positive substances (those that produce an SI between 1.6 and 1.9) in the LLNA: BrdUELISA to determine if such results might be false positives. This could include
evaluations of peptide reactivity, determination of molecular weight, identification of
results from related chemicals, human studies where ethically and scientifically justified,review of occupational exposures and postmarketing experience or monitoring, or in vitro
testing data. All decision criteria should be reassessed as additional discriminators anddata become available.

ICCVAM Recommendations: Performance Standards

8B

ICCVAM concludes that the ICCVAM-recommended performance standards (ICCVAM 2009a) for
the traditional LLNA can be used to evaluate any future modifications of the LLNA: BrdU-ELISA.
The ICCVAM-recommended performance standards for the traditional LLNA apply to the LLNA:
BrdU-ELISA because the test method is functionally and mechanistically similar to the traditional
LLNA. ICCVAM, in conjunction with ECVAM and JaCVAM, developed the internationallyharmonized test method performance standards for the traditional LLNA (ICCVAM 2009a) toevaluate the performance of LLNA test methods that incorporate specific protocol modifications (e.g.,
procedures to measure lymphocyte proliferation) compared to the traditional LLNA. Thus, uniqueperformance standards for the LLNA: BrdU-ELISA are not proposed at this time.

3.0

Validation Status of the LLNA: BrdU-ELISA Test Method

2B

The ICCVAM BRD for the LLNA: BrdU-ELISA test method (Appendix C) provides acomprehensive review of the current validation status of the LLNA: BrdU-ELISA test method,
including its accuracy and reliability, the substances tested, the rationale for the standardized protocol
used for the validation studies, and all available data supporting its validity. This section provides abrief description and summary of the validation status of the LLNA: BrdU-ELISA test method.

3.1

Test Method Description

9B

Originally developed by Takeyoshi et al. (2001) and refined during an interlaboratory validation
study (Kojima et al. 2008), the purpose of the LLNA: BrdU-ELISA test method is to identify
potential skin sensitizers by quantifying lymphocyte proliferation. Like the traditional LLNA, themagnitude of lymphocyte proliferation measured in the LLNA: BrdU-ELISA correlates with theextent to which sensitization develops after a topical induction exposure to a potential skin-sensitizing
substance.

3.1.1

General Test Method Procedures

17B

The test substance is administered topically on three consecutive days to the ears of mice at aconcentration that provides maximum solubility of the test substance without systemic toxicity and/or
excessive local irritation. Two days after the final application of the test substance, 10 mg/mL BrdU,
a thymidine analog, in 0.5 mL physiological saline is administered via intraperitoneal injection toeach mouse. Approximately 24 hours later, the draining auricular lymph nodes are excised, and asingle-cell suspension from the lymph nodes of each animal is prepared for quantifying theincorporation of BrdU, which correlates with lymph node cell proliferation.
The incorporation of BrdU for each mouse is measured using an ELISA and is expressed inabsorbance units. The SI is calculated as the ratio of the mean absorbance/mouse for each treatmentgroup against the mean absorbance/mouse for the vehicle control group. Substances producing an SI
greater than a specified threshold are considered to be sensitizers. Based on the accuracy evaluation
described in Section 3.4, the optimum accuracy was produced by SI ≥ 1.6.

3.1.2

Similarities and Differences Between the Protocols for the Traditional LLNA
and the LLNA: BrdU-ELISA

18B

The differences between the traditional LLNA (Dean et al. 2001; Sailstad et al. 2001; ICCVAM
1999) and the LLNA: BrdU-ELISA include the marker used to detect lymphocyte proliferation, theroute of administration of the marker, and time of lymph node excision. In the traditional LLNA, aradioactive marker such as 3H-methyl thymidine or 125I-iododeoxyuridine (in phosphate-bufferedsaline; 250 µL/mouse) is administered via the tail vein. Then, five hours later, the draining auricularlymph nodes are excised and prepared for quantifying the incorporation of radioactivity. As noted
above, in the LLNA: BrdU-ELISA, a BrdU solution is injected intraperitoneally to each mouse, andthe draining auricular lymph nodes are excised 24 hrs later. All other procedures for the two methods
are identical.

3.2

Validation Database

10B

The current validation database for the LLNA: BrdU-ELISA includes results from studies of
43 substances that had previously been tested in the traditional LLNA. These results were obtained
from six published studies (Takeyoshi et al. 2003; 2004a; 2004b; 2005; 2006; 2007a), several
unpublished studies (Takeyoshi M, unpublished data), one platform presentation (Takeyoshi 2007b),
and one poster presentation (Kojima et al. 2008). The data from Takeyoshi et al. were generated in a

single laboratory while the data from Kojima et al. were generated in multiple laboratories during aninterlaboratory validation study. Data for 31 substances were available and reviewed by the
independent peer review panel in April 2009. Data for 12 additional substances and additional results
for four previously tested substances were submitted after the Panel review. ICCVAM and the OECD
Expert Consultation considered these additional data and the LLNA: BrdU-ELISA BRD was updated
to include the additional data.
The reference test data for the 43 substances were obtained from traditional LLNA tests. Of the
43 substances, 32 were classified by the traditional LLNA as skin sensitizers and 11 were classified as
nonsensitizers. GP skin sensitization data were available for 35 substances and human skinsensitization test data or clinical case report information was available for 41 substances (seeAppendix C, Annex III-1).
Table 3-1 lists the 43 substances, uses, chemical classifications, traditional LLNA EC3 andmaximum stimulation index (SI) values, and LLNA: BrdU-ELISA EC1.6 and maximum SI values.
Nineteen chemical classes were represented by the substances tested in the LLNA: BrdU-ELISA;
11 substances were classified in more than one chemical class. The classes with the highest number ofsubstances were carboxylic acids (13 substances) and aldehydes (six substances). Of the 22 chemicalclasses represented in the NICEATM LLNA database by at least five substances (thereby providing asufficiently large representation for further analyses), 20 classes had at least 60% of the traditional
LLNA results identified as positive. For this database of more than 600 substances, these classes wereidentified as those most likely to be associated with skin sensitization. Fifteen of these classes werealso represented in the LLNA: BrdU-ELISA database (only amides, ethers, ketones, macromolecular
substances, and polycyclic compounds were not included). Among the chemical classes that havebeen previously identified as common skin allergens (e.g., aldehydes, ketones, quinones, andacrylates, [Gerberick et al. 2004]), only ketones were not included in the LLNA: BrdU-ELISA
database. Nevertheless, the Panel considered the database of substances tested in the LLNA: BrdUELISA to be representative of a sufficient range of chemicals typically tested for skin sensitization
potential. The traditional LLNA EC3 values (i.e., estimated concentration needed to produce SI = 3)
for the 33 sensitizers ranged from 0.009% to 47.5%.
Physicochemical characteristics for the 43 substances are provided in Appendix C, Annex II.
Molecular weights ranged from 30.03 to 388.29 g/mole. Twenty-five substances are liquids and
18 substances are solids. Log octanol: water partition coefficients, which were available for41 substances, ranged from -3 to 3.88. Peptide reactivity, which was available for 22 substances,
ranged from high to minimal (Gerberick et al. 2007).

Table 3-1

Product Use and Chemical Classification, Traditional LLNA EC3 Values, LLNA: BrdU-ELISA EC1.6 Values, andMaximum SI Values for 43 Tested Substances
Product Use1

Chemical Class2

Traditional
LLNA EC3(Maximum SI)3

LLNA: BrdUELISA EC1.6(Maximum SI)3

Cosmetics; Manufacturing; Pesticides

Sulfur Compounds;
Heterocyclic Compounds

0.009 (27.7)

0.065 (4.8)

Manufacturing; Pesticides; Pharmaceuticals

Quinones

0.010 (52.3)

0.150 (6.9)

2,4-Dinitrochlorobenzene*

Manufacturing; Pesticides

Hydrocarbon, Halogenated;
Nitro Compounds;
Hydrocarbons, Cyclic

0.049 (43.9)

0.032 (18.8)

Diphenylcyclopropenone

Pharmaceuticals

Hydrocarbons, Cyclic

0.050 (NA)

0.450 (19.1)

Aldehydes

0.083 (18.0)

0.115 (28.6)

Amines0.11 (26.4)

0.285 (14.7)

Substance Name
5-Chloro-2-methyl-4isothaizolin-3-one*
p-Benzoquinone

Glutaraldehyde
4-Phenylenediamine*

Cosmetics; Disinfectant; Manufacturing;
Pesticides
Intermediate in chemical synthesis;
Manufacturing

Formaldehyde

Disinfectant; Manufacturing

Aldehydes

0.50 (4.0)

0.163 (16.6)

Cobalt chloride*

Manufacturing; Pesticides

Inorganic Chemical,
Elements; Inorganic
Chemical, Metals

0.66 (7.2)

0.316 (3.7)

4-Methylaminophenol
sulfateManufacturing

Amines; Phenols

0.8 (6.7)

1.081 (4.0)

trans-Cinnamaldehyde

Food additive; Fragrance agent

Aldehydes

1.4 (13.1)

1.530 (5.9)

Isoeugenol*

Food additive; Fragrance agent

Carboxylic Acids

1.5 (31.0)

5.156 (8.4)

Manufacturing; Pesticides

Heterocyclic Compounds

1.7 (8.6)

12.097 (1.6)

Cosmetics; Food additive; Fragrance agent;Intermediate in chemical synthesis;
Personal care products; Pesticides

Aldehydes

1.9 (18.4)

4.808 (4.0)

Cosmetics; Pharmaceuticals

Amines; Phenols

3.2 (5.7)

2.990 (3.1)

2-Mercaptobenzothiazole*
Cinnamic aldehyde
3-Aminophenol

Product Use1

Chemical Class2

Traditional
LLNA EC3(Maximum SI)3

LLNA: BrdUELISA EC1.6(Maximum SI)3

Food additive; Intermediate in chemical
synthesis

Carboxylic Acids

3.6 (22.6)

8.049 (6.3)

4.7 (4.6)

0.862 (7.9)

4.8 (3.1)

1.027 (4.5)

Amines9.00 (3.3)

11.029 (2.5)

Alcohols; Sulfur
Compounds; Lipids

8.1 (8.9)

13.334 (2.6)

Fragrance agent

Hydrocarbons, Other

9.2 (20.5)

7.143 (16.4)

Food additive; Fragrance agent

Aldehydes

9.7 (20.0)

12.920 (13.5)

Cosmetics; Food additive; Intermediate inchemical synthesis; Manufacturing;
Personal care products; Pharmaceuticals

Carboxylic Acids

10.1 (17.0)

8.851 (17.7)

Phenyl benzoate*

Manufacturing; Pesticides

Carboxylic Acids

13.6 (11.1)

16.954 (3.4)

Cinnamic alcohol*

Cosmetics; Food additive; Fragrance agent;Intermediate in chemical synthesis;
Personal care products

Alcohols

21.0 (5.7)

24.091 (2.7)

Cyclamen aldehyde

Food additive; Fragrance agent

Aldehydes

22.3 (5.2)

41.496 (5.7)

Hydrocarbons, Other

24.0 (8.5)

13.636 (4.8)

Urea

24.0 (5.5)

49.545 (1.6)

Manufacturing

Carboxylic Acids

28.0 (7.0)

31.751 (3.1)

Cosmetics; Food additive; Fragrance agent;
Personal care products; Pesticides

Hydrocarbons, Other

30.0 (8.3)

27.596 (4.7)

Substance Name
Diethyl maleate
Trimellitic anhydride

Manufacturing

Nickel sulfate

Manufacturing

4-Chloroaniline
Sodium lauryl sulfate*
Citral*Hexyl cinnamic aldehyde**

EugenolHydroxycitronellal
Imidazolidinyl urea*
Ethylene glycol
dimethacrylate*
LinaloolIntermediate in chemical synthesis;
Manufacturing; Pesticides; Pharmaceuticals
Cosmetics; Food additive; Manufacturing;
Personal care products; Pesticides;
Pharmaceuticals

Food additive; Fragrance agent; Personalcare products
Cosmetics; Personal care products;
Pesticides

Anhydrides; Carboxylic
Acids
Inorganic Chemicals,
Metals; Inorganic
Chemicals, Elements

Product Use1

Chemical Class2

Traditional
LLNA EC3(Maximum SI)3

LLNA: BrdUELISA EC1.6(Maximum SI)3

Manufacturing

Carboxylic Acids

32.8 (4.0)

33.333 (5.0)

Lipids44.0 (3.4)

9.404 (4.2)

Amines47.5 (4.4)

73.596 (2.1)

Carboxylic Acids

NC (1.3)

NC (1.1)

Carboxylic Acids

NC (1.5)

NC (0.9)

Manufacturing; Fragrance agent

Carboxylic Acids

NC (1.0)

NC (1.3)

GlycerolCosmetics; Food additive; Intermediate inchemical synthesis; Manufacturing;
Personal care products; Pharmaceuticals;Solvent

Alcohols; Carbohydrates

NC (1.1)

NC (1.3)

HexaneManufacturing; Solvent

Hydrocarbons, Acyclic

NC (2.2)

56.328 (1.9)

Alcohols

NC (1.7)

5.344 (2.2)4

Carboxylic Acids

NC (2.2)

15.177 (2.5)

Carboxylic Acids

NC (2.9)

NC (1.4)

Phenols; Carboxylic Acids

NC (2.5)

NC (1.3)

Substance Name
Ethyl acrylate
Isopropyl myristate
Aniline2-Hydroxypropyl
methacrylate
Diethyl phthalate
Dimethyl isophthalate*

Isopropanol
Lactic acid*

Methyl salicylate*
Salicylic acid*

Cosmetics; Personal care products;
Pharmaceuticals
Food additive; Manufacturing; Personalcare products; Pesticides; Pharmaceuticals
Intermediate in chemical synthesis;
Manufacturing
Cosmetics; Manufacturing; Personal careproducts; Pesticides; Pharmaceuticals

Cosmetics; Disinfectant; Food additive;
Intermediate in chemical synthesis;
Manufacturing; Personal care products;
Pharmaceuticals; Solvent
Food additive; Manufacturing;
Pharmaceuticals
Cosmetics; Food additive; Fragrance agent;
Personal care products; Pharmaceuticals;Solvent
Food additive; Manufacturing;
Pharmaceuticals

Sulfanilamide

Pharmaceuticals

Hydrocarbons, Cyclic;
Sulfur Compounds

NC (1.0)

NC (1.3)

Propylene glycol

Cosmetics; Food additive; Intermediate inchemical synthesis; Personal care products;
Pharmaceuticals; Solvent

Alcohols

NC (1.6)

NC (1.6)

Abbreviations: EC3 = estimated concentration (expressed as percentage) needed to produce SI = 3; EC1.6 = estimated concentration (expressed as percentage)needed to produce SI = 1.6; LLNA = murine local lymph node assay; LLNA: BrdU-ELISA= local lymph node assay with enzyme-linked immunosorbent
assay detection of bromodeoxyuridine; NA = not available; NC = not calculated since maximum SI < 3.0 for the traditional LLNA or maximum SI < 1.6 forthe LLNA: BrdU-ELISA; SI = stimulation index.
* Reference substance from ICCVAM (2009a).
1

Information gathered from the following databases:
Hazardous Substances Database (http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB)
Haz-Map (http://hazmap.nlm.nih.gov/)
Household Products Database (http://hpd.nlm.nih.gov/index.htm)
International Programme on Chemical Safety INCHEM database (http://www.inchem.org/)
National Toxicology Program (http://ntp.niehs.nih.gov:8080/index.html?col=010stat).

2

Chemical classifications based on the Medical Subject Headings classification for chemicals and drugs, developed by the National Library of Medicine(http://www.nlm.nih.gov/mesh/meshhome.html).

3

Mean EC3 (expressed as percent concentration) and maximum SI values are from the NICEATM database of traditional LLNA studies. EC1.6 and SI values
for individual LLNA: BrdU-ELISA tests are provided in Annex IV of the BRD (Appendix C).

4

Highest SI of seven tests. Because the majority (five) of the seven tests, had SI values < 1.6, isopropanol is considered to be a nonsensitizer in the LLNA:
BrdU-ELISA.

3.3

Reference Test Method Data

11B

Thirty-five of the 43 substances that were tested in the traditional LLNA were considered in theoriginal evaluation of the LLNA by ICCVAM (ICCVAM 1999). The traditional LLNA reference data
used for the accuracy evaluation were obtained from ICCVAM (1999) for 33 of these substances.
Data for two substances which were negative in the original LLNA evaluation (ICCVAM 1999),
aniline and nickel sulfate, were obtained from more recent sources that tested higher concentrations
and obtained positive results. The traditional LLNA data for the remaining eight substances that were
not considered in the original ICCVAM evaluation were obtained from the scientific literature. The
reference data for GP tests (GPMT or Buehler test) and human tests (human maximization test,
human patch test allergen, or other human data) were also obtained from the original LLNA
evaluation (ICCVAM 1999) and the scientific literature. The LLNA, GP, and human reference dataand sources for the 43 substances evaluated are provided in Annex III of the BRD (Appendix C).

3.4

Test Method Accuracy

12B

The ICCVAM evaluation of the LLNA: BrdU-ELISA included an assessment of multiple decision
criteria including SI ≥ 2.0, the threshold for distinguishing sensitizers and nonsensitizers that wasused in the protocol for the interlaboratory validation study (Kojima et al. 2008) (Table 3-2). Whenthe optimal decision criterion of SI ≥ 1.6 was used to identify sensitizers vs. nonsensitizers, comparedto the traditional LLNA, accuracy was 95% (41/43), with a false positive rate of 18% (2/11) and afalse negative rate of 0% (0/32). The two false positive substances, hexane (SI = 1.76 and 1.89) andlactic acid (SI = 1.80, 1.89, and 2.53), produced SI values between 1.6 and 1.9 in the LLNA: BrdUELISA. Other available information such as dose-response, evidence of systemic toxicity or excessive
local irritation, and (where appropriate) statistical significance together with SI values should beconsidered to confirm that such borderline results are potential skin sensitizers. Consideration should
also be given to various properties of the test substance, including whether it is structurally similar to
known skin sensitizers. For example, peptide reactivity (Gerberick et al. 2007) could be used tointerpret LLNA: BrdU-ELISA results when borderline positive results (e.g., SI values between 1.6
and 1.9) are produced to confirm that such results are not false positive. Both of the LLNA
nonsensitizers with positive results in the LLNA: BrdU-ELISA, lactic acid and hexane, had minimal
peptide reactivity. No unique characteristics were identified that could be used as rationale for
excluding any particular types of substances from testing in the LLNA: BrdU-ELISA.
An evaluation to determine the robustness of the optimum SI ≥ 1.6 criterion indicated that the SI wasquite stable. Taking different samples of the data as training and validation sets had relatively little
impact on the cutoff SI criteria or on the resulting number of false positives or false negatives(Appendix C, Annex VII).
Figure 3-1 shows that SI values for the LLNA: BrdU-ELISA are generally lower than those for the
traditional LLNA at comparable test doses. SI values for substances with more than one test result are
represented by the geometric mean with bars to show the overall range of individual study results
used to calculate the geometric mean. The purpose of showing the geometric mean and associatedranges is to provide an assessment of variability among results, and the relative sensitivity of the
traditional LLNA and LLNA: BrdU-ELISA results. However, the accuracy analyses reported in the
BRD are based on individual test results and not on a geometric mean. The SI values for Figure 3-1
are provided in Table 3-3.

Table 3-2

Alternate
Criterion

Performance of the LLNA: BrdU-ELISA for 43 Substances in Predicting Skin Sensitizing Potential Using Alternative
Decision Criteria to Identify Sensitizers
Accuracy

Sensitivity

Specificity

False Positive
Rate

False Negative
Rate

Positive
Predictivity

Negative
Predictivity%(No.1)%(No. 1)%(No. 1)%(No. 1)%(No. 1)%(No. 1)%(No. 1)

Statistics2

91(39/43)

97(31/32)73(8/11)

27(3/11)

3(1/32)

91(31/34)89(8/9)≥95% CI3

88(38/43)100(32/32)54(6/11)

46(5/11)

0(0/32)

86(32/37)

100

(6/6)

≥2 SD491(39/43)100(32/32)64(7/11)

36(4/11)

0(0/32)

89(32/36)

100

(7/7)

≥3 SD591(39/43)91(29/32)

91(10/11)9(1/11)

9(3/32)

97(29/30)77(10/13)SI ≥ 5.0

49(21/43)31(10/32)100(11/11)0(0/11)

69(22/32)100(10/10)33(11/33)SI ≥ 4.5

58(25/43)44(14/32)100(11/11)0(0/11)

56(18/32)100(14/14)38(11/29)SI ≥ 4.0

63(27/43)50(16/32)100(11/11)0(0/11)

50(16/32)100(16/16)41(11/27)

SI ≥ 3.5

74(32/43)

66(21/32)100(11/11)0(0/11)

34(11/32)100(21/21)50(11/22)SI ≥ 3.084(36/43)78(25/32)100(11/11)0(0/11)

22(7/32)

100(25/25)61(11/18)SI ≥ 2.5

93(40/43)91(29/32)100(11/11)0(0/11)

9(3/32)

100(29/29)79(11/14)SI ≥ 2.0

95(41/43)

94(30/32)100(11/11)0(0/11)

6(2/32)

100(30/30)85(11/13)SI ≥ 1.9

95(41/43)

94(30/32)100(11/11)0(0/11)

6(2/32)

100(30/30)85(11/13)SI ≥ 1.6

95(41/43)100(32/32)82(9/11)

18(2/11)

0(0/32)

94(30/32)

100

(9/9)

SI ≥ 1.5

95(41/43)100(32/32)82(9/11)

18(2/11)

0(0/32)

94(30/32)

100

(9/9)

SI ≥ 1.3

93(40/43)100(32/32)73(8/11)

27(3/11)

0(0/32)

91(32/35)

100

(8/8)

Abbreviations: CI = confidence interval; LLNA: BrdU-ELISA = murine local lymph node assay with enzyme-linked immunosorbent assay detection of
bromodeoxyuridine (BrdU); No. = number; SD = standard deviation; SI = stimulation index
1

The proportion on which the percentage calculation is based.

2

Analysis of variance for difference of group means when substances were tested at multiple doses or t-test when substances were tested at one dose. The
absorbance data were log-transformed prior to analysis of variance. Significance at p < 0.05 was further tested by Dunnett’s test.

3

The mean absorbance of at least one treatment group was outside the 95% confidence interval for the mean absorbance of the vehicle control group.

4

The mean absorbance of at least one treatment group was greater than 3 SD from the mean absorbance of the vehicle control group.

5

The mean absorbance of at least one treatment group was greater than 2 SD from the mean absorbance of the vehicle control group.

Figure 3-1

Comparison of LLNA: BrdU-ELISA Stimulation Index with Traditional
LLNA Stimulation Index1

Abbreviations: CMI = 5-chloro-2-methyl-4-isothiazoline-3-one solution; DPCP = diphenylcyclopropanone;
DNCB = 2,4-dinitrochlorobenzene; EGDA = ethylene glycol dimethacrylate; False + = false positive
results in the LLNA: BrdU-ELISA (based on most prevalent result for substances with multiple tests)
were in the SI range between 1.6 and 1.9; HCA = hexyl cinnamic aldehyde; HPMA = 2-hydroxypropyl
methacrylate; LLNA = murine local lymph node assay; LLNA: BrdU-ELISA = murine local lymph node
assay with enzyme-linked immunosorbent assay detection of bromodeoxyuridine; MAPS = 4-methyl
aminophenol sulfate; MBT = 2-mercaptobenzothiazole; Ni = nickel; SI = stimulation index.
1

LLNA: BrdU-ELISA and traditional LLNA responses at comparable test doses are shown. Symbols
show the SI for substances with one test result or geometric mean maximum SI for substances with morethan one test result. Table 3-3 shows the individual values used. Bars show the range of values reported
for multiple test results (heavy bars for LLNA: BrdU-ELISA and light bars for traditional LLNA).
Numbers in parentheses beside the chemical names show the number of SI values for the LLNA: BrdUELISA and then the number of SI values for the traditional LLNA used in this figure. The number of SI
values used in the figure may be different from the total number of SI values available since only
comparable test doses and vehicles were used in this figure. The accuracy analyses used individual test
results rather than geometric mean SI values. Using individual test results, traditional LLNA
nonsensitizers with maximum SI between 1.6 and 1.9 include hexane and lactic acid.

* The LLNA: BrdU-ELISA SI for diethyl phthalate is outside of the displayed data range and is not shown(SI < 1).

Table 3-3

Maximum SI Values of 43 Substances Evaluated in the LLNA: BrdU-ELISA
Compared to the Traditional LLNA

Substance Name

1

Test
Vehicle2

LLNA: BrdU-ELISA
Maximum SI Values3

Traditional LLNA
Maximum SI Values

Sensitizers (LLNA: BrdU-ELISA SI ≥ 1.6 and Traditional LLNA SI ≥ 3.0)
Benzoquinone (1,1)
1,4-Phenylenediamine (1,3)
Diphenylcyclopropenone(1,1)
2,4-Dinitrochlorobenzene(7,5)
CMI (1,1)
Diethyl maleate (1,1)
Glutaraldehyde (3,1)

AOO
AOO

6.94
14.70

52.30
23.30, 37.40, 75.30

AOO/ACE

19.10

31.70

3.68, 4.50, 5.29, 6.26,
6.53, 12.30, 18.80
4.83
6.27
2.25, 3.72, 28.60

23.00, 24.00, 26.80,
36.70, 49.60
22.70
22.60
18.00
10.00, 11.60, 11.60,
13.40, 14.00, 14.00,
14.10, 14.50, 16.00,
17.00, 17.00, 17.00,
17.60, 20.00
13.10
7.60, 15.80, 18.40
4.01, 6.10, 9.30, 9.60,
10.20, 12.40, 14.10,
16.00, 16.10, 16.10,
17.00, 70.30
4.10, 4.90, 5.00, 5.60,
6.70, 6.80, 7.20, 7.20,
7.50, 7.50, 7.60, 8.70,
10.00, 11.00, 11.10,
11.80, 12.40, 13.80,
13.10, 13.10, 13.10,
14.10, 14.70, 14.70,
15.30, 17.00, 18.40,
19.00, 23.20, 19.20,
19.30, 23.20, 23.60,
24.40, 29.80, 31.00
4.60, 9.10, 9.50, 10.80,
17.10
4.70, 6.20, 9.30, 20.50
8.50
8.30
7.21
7.00
6.70
3.50, 11.10
5.70
continued

AOO
DMF
AOO
ACE

HCA (11,14)

AOO

2.72, 2.87, 3.02, 3.27,
3.34, 3.40, 3.60, 3.64,
3.84, 5.90, 13.50

trans-Cinnamaldehyde (4,1)
Cinnamic aldehyde (1,3)

AOO
AOO

3.37, 3.50, 4.11, 5.86
3.97

Eugenol (6,12)

AOO

3.05, 3.17, 3.18, 7.09,
12.30, 17.70

Isoeugenol (4,36)

AOO

2.36, 2.43, 7.20, 8.36

MBT (1,5)

DMF

1.62

AOO
AOO
AOO
DMSO
MEK
DMF
DMF/AOO
AOO

16.40
1.34, 4.78
1.45, 4.65
3.68
3.11
3.98
3.37
3.06

Citral (1,4)
Hydroxycitronellal (2,1)
Linalool (2,1)
Cobalt chloride (1,1)
EGDA (1,1)
MAPS (1,1)
Phenyl benzoate (1,2)
3-Aminophenol (1,1)

Table 3-3

Maximum SI Values of 43 Substances Evaluated in the LLNA: BrdU-ELISA
Compared to the Traditional LLNA (continued)

Substance Name

1

Test
Vehicle2

LLNA: BrdU-ELISA
Maximum SI Values3

Traditional LLNA
Maximum SI Values

Sensitizers (LLNA: BrdU-ELISA SI ≥ 1.6 and Traditional LLNA SI ≥ 3.0)
Imidazolidinyl urea (1,1)
Cyclamen aldehyde (1,1)
Trimellitic anhydride (1,1)

DMF
AOO
AOO

1.61
1.97, 5.71
7.85

Sodium lauryl sulfate (1,7)

DMF

2.64

Formaldehyde (3, 1)
Ethyl acrylate (1,1)
Cinnamic alcohol (1,1)
Isopropyl myristate (1,1)
Ni sulfate (3,1)
Aniline (1,2)
4-Chloroaniline (1,4)

5.50
5.16
4.60
1.60, 2.60, 4.10, 5.10,
5.10, 5.40, 8.90
4.00
3.98
3.90
3.40
3.10
1.70, 3.30
1.80, 1.80, 2.50, 3.30

ACE
1.97, 4.40, 16.60
AOO
4.95
AOO
2.74
AOO
4.19
DMSO
2.58, 2.66, 4.53
AOO
2.07
AOO
2.53
Traditional LLNA Nonsensitizers (SI <3.0)with Borderline Positive SI Values in LLNA: BrdU-ELISA (1.6 < SI < 1.9; see bold text)
Hexane (1,1)
AOO
1.38, 1.89
2.20
Lactic acid (3,1)
DMSO
1.80, 1.89, 2.53
2.20
Nonsensitizers (LLNA: BrdU-ELISA SI < 1.6 and Traditional LLNA SI < 3.0)
Salicylic acid (1,1)

AOO

1.26

Methyl salicylate (3,7)

AOO

1.40, 1.44, 1.44

Isopropanol (6,1)

AOO

Propylene glycol (2,1)
Diethyl phthalate (1,1)
HPMA (1,1)
Glycerol (1,1)
Dimethyl isophthalate (1,1)
Sulfanilamide (1,1)

AOO/Water
AOO
AOO
Water/DMF
AOO
DMF

0.94, 0.98, 1.01, 1.57,
2.04, 2.22
0.87, 1.57
0.88
1.13
1.29
1.26
1.26

2.50
0.90, 1.10, 1.72, 1.90,
2.10, 2.30, 2.90
1.70
1.60
1.50
1.30
1.10
1.00
1.00

Abbreviations: ACE = acetone; AOO = acetone: olive oil (4:1); CMI = 5-Chloro-2-methyl-4-isothiazoline3-one solution; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; EGDA = ethylene glycol
dimethacrylate; HCA = hexyl cinnamic aldehyde; HPMA = 2-hydroxypropyl methacrylate;
LLNA = murine local lymph node assay; LLNA: BrdU-ELISA = murine local lymph node assay withenzyme-linked immunosorbent assay detection of bromodeoxyuridine; MAPS = 4-methyl aminophenol
sulfate; MBT = 2-mercaptobenzothiazole; Ni sulfate = nickel (II) sulfate hexahydrate; SI = stimulationindex.
1

Numbers in parentheses beside the substance names indicate the number of tests for the LLNA: BrdUELISA followed by the traditional LLNA, which may differ from the total number of tests available since
only the most comparable test doses and vehicles were included.

2

The vehicle used was the same in LLNA: BrdU-ELISA and traditional LLNA tests, except whereindicated (e.g., vehicle used in the LLNA: BrdU-ELISA/vehicle used in the traditional LLNA).

3

The bold text indicates SI values having potential false positive results (1.6 < SI < 1.9) for individual
LLNA: BrdU-ELISA tests

3.5

Test Method Reliability (Intra- and Interlaboratory Reproducibility)

13B

The BRD details the evaluation of intra- and interlaboratory reproducibility of the LLNA: BrdUELISA test method. Intralaboratory reproducibility was assessed using a concordance analysis ofsensitizer/nonsensitizer results, and a coefficient of variation (CV) analysis of SI values andEC1.6 values (estimated concentration needed to produce an SI of 1.6). The qualitative analysis
shows that multiple tests of 12 substances (10 LLNA sensitizers and two nonsensitizers) yielded
100% concordance for sensitizer/nonsensitizer outcomes for 83% (10/12) of the substances. The
concordant results for one nonsensitizer, hexane, however, were incorrectly positive for both tests(2/2 tests had SI ≥ 1.6). In the quantitative analyses, the CVs for the SI values of 13
substance/concentration combinations that were tested up to five times each ranged from 1% to80%. In addition, the CVs for the EC1.6 values of four substances that were tested up to fivetimes at multiple doses ranged from 37% to 118%.
When using SI ≥ 1.6 as the threshold to distinguish sensitizers from nonsensitizers, the qualitativeinterlaboratory reproducibility analysis of 10 substances (seven sensitizers and three
nonsensitizers) that were tested in three to seven laboratories indicated 100% agreement (3/3, 6/6,
or 7/7) among the laboratories for nine substances (seven sensitizers and two nonsensitizers).
However, one of the nonsensitizers, lactic acid, for which there was 100% agreement among thelaboratories, was a false positive (i.e., 3/3 laboratories had SI ≥ 1.6). There was 67% (4/6)
agreement among the tests for the remaining nonsensitizer. Interlaboratory CVs for the EC1.6
values of the seven sensitizers ranged from 31% to 93%.
When using SI ≥ 1.6 to classify sensitizers, the concordance analysis for the 18 substances withmultiple tests indicated that the SI results for 85% (11/13) of the sensitizers (based on traditional
LLNA results) were 100% concordant (i.e., all tests yielded SI ≥ 1.6) (Table 3-4). The SI results
for the remaining two sensitizers included one test with SI < 1.6 and another test with SI > 1.6.
The SI results for 60% (3/5) of the nonsensitizers were 100% concordant. All tests for two of thethree nonsensitizers yielded SI < 1.6. All tests for the third nonsensitizer yielded SI values
between 1.6 and 1.9, the narrow region in which false positive results occurred. The concordance
for the other two nonsensitizers was 71% (5/7) for SI < 1.6 and 67% (2/3) for SI values between
1.6 and 1.9.
Table 3-4

Concordance of LLNA: BrdU-ELISA Tests across Maximum SI Categories

Substance
Sensitizers2
Cyclamen aldehyde
2,4-Dinitrochlorobenzene
Diphenylcyclopropenone
EugenolFormaldehyde
Glutaraldehyde

LLNA: BrdUELISA
Nonsensitizers(MaximumSI ≤ 1.61)

LLNA: BrdU-ELISA Sensitizers(Maximum SI ≥ 1.6)

Total
Tests

1.6 < Maximum SI< 1.91

Maximum
SI ≥ 1.91

0 (0%)

0 (0%)

0 (100%)2

0 (0%)

0 (0%)

9 (100%)90 (0%)

0 (0%)

3 (100%)30 (0%)
0 (0%)
0 (0%)

0 (0%)
0 (0%)
0 (0%)

9 (100%)3 (100%)5 (100%)935continued

Table 3-4

Concordance of LLNA: BrdU-ELISA Tests across Maximum SI Categories(continued)

Substance
Sensitizers2
Hexyl cinnamic
aldehyde
Hydroxycitronellal
Isoeugenol
Linalool
Nickel sulfate
1,4-Phenylenediaminetrans-Cinnamaldehyde
Nonsensitizers2
HexaneIsopropanol
Lactic acid
Methyl salicylate
Propylene glycol

LLNA: BrdUELISA
Nonsensitizers(MaximumSI ≤ 1.61)

LLNA: BrdU-ELISA Sensitizers(Maximum SI ≥ 1.6)

Total
Tests

1.6 < Maximum SI< 1.91

Maximum
SI ≥ 1.91

0 (0%)

0 (0%)

12 (100%)12

1 (50%)
0 (0%)
1 (50%)
0 (0%)
0 (0%)
0 (0%)

0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)

1 (50%)
3 (100%)1 (50%)
3 (100%)2 (100%)4 (100%)

2
3
2
3
2
4

0 (0%)
5 (71%)
0 (0%)
3 (100%)3 (100%)2 (100%)0 (0%)
2 (67%)
0 (0%)
0 (0%)0 (%)
2 (29%)
1 (33%)
0 (0%)
0 (0%)

27
3
3
3

Abbreviations: LLNA: BrdU-ELISA = murine local lymph node assay with enzyme-linked immunosorbent
assay detection of bromodeoxyuridine; SI = stimulation index.
1

Numbers shown reflect number of tests. Percentage in parentheses reflects percentage of the total numberof tests for each substance.

2

According to traditional murine local lymph node assay results.

3.6

Animal Welfare Considerations: Reduction, Refinement, and Replacement

14B

The LLNA: BrdU-ELISA will use the same number of animals as the updated ICCVAMrecommended traditional LLNA protocol (Appendix A of ICCVAM 2009a). However, since useof the traditional LLNA is restricted in some countries and institutions because of limitations on
handling radioactivity, availability and use of the nonradioactive LLNA: BrdU-ELISA may leadto further reduction in use of the GP tests, which would provide for reduced animal use andincreased refinement due to the avoidance of pain and distress that occur in the GP tests whensubstances cause ACD. Additionally, the LLNA: BrdU-ELISA test method protocol requires
fewer mice per treatment group (a minimum of four animals/group) than either of the GP tests(10-20 animals/group for the Buehler test and 5-10 animals/group for the GPMT).

4.0

ICCVAM Consideration of Independent Peer Review Panel
Report and Other Comments
3B

The ICCVAM evaluation process incorporates a high level of scientific peer review andtransparency. The evaluation process for the LLNA: BrdU-ELISA included two public review
meetings by an independent scientific peer review panel, multiple opportunities for publiccomments (see Section 1.0), consideration of the OECD Expert Consultation on the LLNA, andcomments from the SACATM. ICCVAM and the IWG considered the Panel report, conclusionsof the OECD Expert Consultation, the SACATM comments, and all public comments before
finalizing the ICCVAM Test Method Evaluation Report and final BRD for the LLNA: BrdUELISA. This chapter summarizes the ICCVAM consideration of these reports and comments. The
peer review panel reports and public comments are provided as Appendices D and E,
respectively. The report of the OECD Expert Consultation on the LLNA is not publicly available.

4.1

ICCVAM Consideration of Independent Peer Review Panel Report andOECD Comments

4.1.1

Comments on Revised Draft ICCVAM Recommendations: Test Method
Usefulness and Limitations

15B

19B

The Panel agreed that the available data and test method performance supported the use of the
LLNA: BrdU-ELISA to identify substances as potential sensitizers and nonsensitizers, withcertain limitations. The Panel noted that the accuracy analysis they reviewed supported using two
decision criteria (i.e., one to identify sensitizers and one to identify nonsensitizers). The Panelemphasized that the decision criteria were empirically derived from the data and produced thebest combination of maximum accuracy coupled with the minimum number of results in therange of uncertainty (i.e., the range in which maximum SI results were between the decision
criteria for sensitizers and nonsensitizers). Since using two decision criteria allows for a moredefinitive identification of sensitizers and nonsensitizers, this approach provides animal welfare
benefits by reducing further tests that might be required in instances where the hazard
classification of a substance is not as clear. In addition, one can use statistical analysis and/or
other data and information (e.g., peptide reactivity, quantitative structure-activity relationships,
skin penetration information) to provide more information on compounds that fall in the range of
uncertainty. However, the Panel questioned how results in the range of uncertainty would be
useful for regulatory purposes and emphasized that additional guidance would be needed on how
to classify substances with SI values in the range of uncertainty.
The OECD LLNA Expert Consultation viewed that despite certain limitations, the LLNA: BrdUELISA is useful as a modified LLNA test method that has the potential to reduce the number ofanimals required and refine the way in which animals are used for ACD testing. The experts
reviewed LLNA: BrdU-ELISA results for 12 additional substances and four substances
previously tested that were received by NICEATM after the Panel meeting. Like the Panel,
OECD member country experts questioned the regulatory utility of the LLNA: BrdU-ELISA
since specific guidance on how to classify substances with SI values in the range of uncertainty
had not been developed. Therefore, they recommended instead that a single decision criterion (aswas originally proposed by ICCVAM and reviewed by the Panel in 2008) would be more useful
to identify substances as potential sensitizers. They agreed with ICCVAM that SI ≥ 1.6 provided
the optimal test method performance by preventing false negative results. They also agreed withICCVAM that users may want to consider additional information such as dose-response, evidenceof systemic toxicity and/or excessive local skin irritation, and where appropriate, statisticalsignificance together with SI values to confirm borderline positive results (i.e., SI between 1.6
and 1.9) as potential skin sensitizers.
ICCVAM considered the Panel report and the OECD Expert Consultation recommendations, andconcluded that the single SI decision criterion of SI ≥ 1.6 to classify sensitizers would avoid false
negative results as well as indeterminate results, which are not useful for regulatory purposes.
Borderline results that may occur between 1.6 and 1.9 could be evaluated using other information
to confirm the result.

4.1.2

Comments on Revised Draft ICCVAM Recommendations: Test Method
Protocol
20B

The Panel concurred with ICCVAM that the validation studies indicated that the standardized
protocol was sufficiently transferable and reproducible. The Panel agreed that laboratories should
maintain a historical database of positive control SI values and some measure of variability overtime. The evaluation of the variation in positive control responses over time has wide
applicability to a broad range of test systems.
The Panel agreed with the ICCVAM-recommended protocol, which indicated that all existing
toxicological information (e.g., acute toxicity and dermal irritation) and structural andphysicochemical information on the test substance of interest (and/or structurally related test
substances) should be considered, where available, in selecting three consecutive doses. The
OECD Expert Consultation also agreed and emphasized that the highest dose should be theconcentration that maximizes exposure while avoiding systemic toxicity and/or excessive local
skin irritation after topical application in the mouse. In the absence of such information, andconsistent with the updated ICCVAM recommended protocol, a prescreen test should be
performed in order to define the appropriate dose level to test in the LLNA: BrdU-ELISA. The
Panel and the OECD Expert Consultation agreed in principle with ICCVAM that use of a reduced
LLNA: BrdU-ELISA test method protocol instead of the multidose LLNA: BrdU-ELISA test
method protocol has the potential to reduce the number of animals used in a test by omitting the
middle and low dose groups. However, some members of the OECD Expert Consultation
speculated that the reduced LLNA would have limited regulatory use and therefore the extent ofpotential animal savings is difficult to estimate.

4.1.3

Comments on the Revised Draft ICCVAM Recommendations: FutureStudies

21B

The Panel concurred with ICCVAM’s revised draft recommendations for future studies,emphasizing that additional decision criteria and guidance should be identified for substances that
produce SI values in the range of uncertainty, and that the additional decision criteria should bereassessed as additional discriminators and data become available (e.g., high-quality human ACDdata). While the range of uncertainty is eliminated when using the single decision criterion ofSI ≥ 1.6, the OECD Expert Consultation recommended that borderline positive results (i.e., SI
values between 1.6 and 1.9) be further evaluated to determine if they are correctly identified as
potential skin sensitizers.
The Panel recommended further consideration of statistical issues, including how to determineand evaluate classification methods (i.e., classification cutoff points). The Panel also
recommended that future interlaboratory validation studies should simultaneously evaluate
intralaboratory reproducibility, using appropriate statistics, to evaluate variation both within alaboratory and between laboratories.
ICCVAM considered the Panel report and the OECD Expert Consultation recommendations andconcluded that efforts should be made to further characterize the sensitization potential of

borderline positive substances that produce an SI between 1.6 and 1.9 in the LLNA: BrdU-ELISA
to confirm that such results are not false positive.

4.1.4

Comments on Revised Draft ICCVAM Recommendations: PerformanceStandards
22B

The Panel agreed that the ICCVAM-recommended LLNA performance standards state theessential test method requirements, and the LLNA: BrdU-ELISA adheres to them such that itshould be considered mechanistically and functionally similar. The only variation with thetraditional LLNA is the means by which lymphocyte proliferation during the induction phase isevaluated. Likewise, the OECD Expert Consultation also considered the LLNA: BrdU-ELISA to
be mechanistically and functionally similar to the LLNA, and therefore agreed that the LLNA
performance standards are applicable.

4.2

ICCVAM Consideration of Public and SACATM Comments

16B

The ICCVAM evaluation process incorporates a high level of transparency. This process isdesigned to provide numerous opportunities for stakeholder involvement, including submitting
written public comments and providing oral comments at ICCVAM independent peer review
panel meetings and SACATM meetings. Table 4-1 lists the 12 different opportunities for publiccomment that were provided during the ICCVAM evaluation of the validation status of newversions and applications of the LLNA. The number of public comments received in response toeach of the opportunities is also indicated. A total of 49 comments were submitted. Comments
received in response to or related to the FR notices are available on the NICEATM-ICCVAM
website. 12 The following sections, delineated by FR notice, briefly discuss the public comments
received.
F

F

Table 4-1

Opportunities for Public Comments
Opportunities for Public Comments

Date

Number of Public
Comments
Received

72 FR 27815: The Murine Local Lymph Node Assay:
Request for Comments, Nominations of Scientific Experts,
and Submission of Data

May 17, 2007

17

72 FR 52130: Draft Performance Standards for the Murine
Local Lymph Node Assay: Request for Comments

September 12, 2007

4

73 FR 1360: Announcement of an Independent ScientificPeer Review Panel Meeting on the Murine Local Lymph
Node Assay; Availability of Draft Background Review
Documents; Request for Comments

January 8, 2008

7

Independent Scientific Peer Review Panel Meeting Assessing
the Allergic Contact Dermatitis Potential of Chemicals andProducts: Validation Status of New Versions andApplications of the Murine Local Lymph Node Assay

March 4-6, 2008

16

73 FR 25754: Meeting of the Scientific Advisory Committee
on Alternative Toxicological Methods (SACATM)

May 7, 2008

1
continued

12

Available at http://ntp-apps.niehs.nih.gov/iccvampb/searchPubCom.cfm

Table 4-1

Opportunities for Public Comment (continued)
Opportunities for Public Comments

Date

Number of Public
Comments
Received

73 FR 29136: Peer Review Panel Report on the Validation
Status of New Versions and Applications of the Murine
Local Lymph Node Assay (LLNA): A Test Method forAssessing the Allergic Contact Dermatitis Potential ofChemicals and Products: Notice of Availability and Request
for Public Comments

May 20, 2008

0

SACATM Meeting, Radisson Hotel, RTP, NC

June 18-19, 2008

074 FR 8974: Announcement of a Second Meeting of the
Independent Scientific Peer Review Panel on the Murine
Local Lymph Node Assay; Availability of Draft Background
Review Documents (BRD); Request for Comments

February 27, 20091

Independent Scientific Peer Review Panel Meeting Assessing
the Allergic Contact Dermatitis Potential of Chemicals andProducts: Evaluation of the Updated Validation Status of
New Versions and Applications of the Murine Local Lymph
Node Assay

April 28-29, 20092

74 FR 19562: Meeting of the Scientific Advisory Committee
on Alternative Toxicological Methods (SACATM)

April 29, 2009

074 FR 26242: Independent Scientific Peer Review Panel
Report: Updated Validation Status of New Versions andApplications of the Murine Local Lymph Node Assay: A
Test Method for Assessing the Allergic Contact Dermatitis
Potential of Chemicals and Products: Notice of Availability
and Request for Public Comments

June 1, 2009

1

SACATM Meeting, Hilton Arlington Hotel, Arlington, VA

June 25-26, 2009

0

4.2.1

Public Comments in Response to 72 FR 27815 (May 17, 2007): The Murine
Local Lymph Node Assay: Request for Comments, Nominations of ScientificExperts, and Submission of Data
23B

NICEATM requested the following:
1. Public comments on the appropriateness and relative priority of evaluation of thevalidation status ofa. The LLNA as a stand-alone assay for determining potency (including severity)
for the purpose of hazard classificationb. The reduced LLNA approach (Kimber et al. 2006; ESAC 2007; ICCVAM
2009b)
c. Nonradioactive LLNA methods
d. The use of the LLNA for testing mixtures, aqueous solutions, and metals
e. The current applicability domain
2. Nominations of expert scientists to consider as members of a possible peer review
panel
3. Submission of data for the LLNA and/or modified versions of the LLNA
In response to this FR notice, NICEATM received 17 comments. Six comments included
additional data and information, while two others offered data and information upon request.

Three commenters nominated four potential panelists for consideration. Three commenterssuggested reference publications for consideration during the Panel evaluation. The nominees
were included in the database of experts from which the Panel was selected. The data andsuggested references were included in the draft ICCVAM review documents that were provided
to the Panel at the March 2008 meeting.
1. A commenter suggested rearranging the priority sequence of test method evaluationfrom most to least pressing: a, e, d, b, and c (see list above).
•

ICCVAM did not establish a relative priority for these activities because they were
all considered to be high-priority activities. Accordingly, all LLNA-related activities
described above were discussed at the March 2008 Panel meeting.

One comment pertained to the LLNA: BrdU-ELISA.
1. One commenter indicated that several nonradioactive detection methods for the
LLNA (e.g., BrdU incorporation, methods measuring the release of various
cytokines, methods using fluorescent markers, and quantification by flow cytometry)
have been developed and shown to be as sensitive as protocols involving
radiolabeling. The commenter indicated that since both ECVAM and JaCVAM werereviewing some of these types of nonradioactive methods that ICCVAM shouldcollaborate with these ongoing efforts rather than initiate a comprehensive
independent review.
•

4.2.2

In 2007, the CPSC requested that ICCVAM evaluate several modifications of the
LLNA, which included the LLNA: BrdU-ELISA. After considering comments fromthe public and the SACATM, ICCVAM assigned the activity a high priority.
Scientists from ECVAM and JaCVAM served as liaisons to the IWG during theevaluation of the LLNA: BrdU-ELISA and actively participated in the review. Bothliaisons nominated scientists to the peer review panel and the JaCVAM liaison
provided much of the validation data for the review.

Public Comments in Response to 72 FR 52130 (September 12, 2007): Draft
Performance Standards for the Murine Local Lymph Node Assay: Request
for Comments
24B

NICEATM requested public comments on the September 2007 draft ICCVAM-recommended
LLNA performance standards developed to facilitate evaluation of modified LLNA test method
protocols with regard to the traditional LLNA. In response to this FR notice, NICEATM received
four comments, two of which suggested clarifications to the text. Another comment
recommended that test substances chosen for testing in the various LLNA methods should be
pure, with conclusive structures, and should not be mixtures. Most comments specificallyaddressed the LLNA performance standards, although one comment pertained to the LLNA ingeneral.
1. One commenter supported the development of performance standards that expedite
the validation of new protocols similar to previously validated methods but wasdisappointed that NICEATM-ICCVAM had chosen to develop performancestandards for such a narrow scope of applicability (i.e., modifications of the standard
LLNA that involve incorporation of nonradioactive methods of detecting lymphocyte
proliferation). The commenter suggested that limited resources available to
NICEATM-ICCVAM would be better spent on activities that would have greater
impact on the reduction, refinement, or replacement of animal use, such as evaluating
the use of human cell lines or in vitro skin models as a replacement for the LLNA.

•ICCVAM considered the comment and concluded that the proposed modifications to
the LLNA test method protocol and expanded applications have the potential tofurther reduce and refine animal use. ICCVAM is committed to identifying in vitro
models and non-animal approaches for assessing ACD and is engaged with ECVAM
and JaCVAM in the development of validation studies for such methods.

There were no comments that specifically addressed the LLNA: BrdU-ELISA.

4.2.3

Public Comments in Response to 73 FR 1360 (January 8, 2008):
Announcement of an Independent Scientific Peer Review Panel Meeting on
the Murine Local Lymph Node Assay; Availability of Draft Background
Review Documents; Request for Comments
25B

NICEATM requested public comments on the January 2008 draft BRDs, draft ICCVAM test
recommendations, draft test method protocols, and updated draft LLNA performance standards
for an international independent scientific peer review panel meeting to evaluate modifications
and new applications for the LLNA. NICEATM received 23 comments in response to this FR
notice; seven written comments were received in advance of the meeting, and 16 oral comments
were offered at the Panel meeting.
Two written comments were relevant to the LLNA: BrdU-ELISA.
1. One commenter noted that the LLNA: BrdU-ELISA was recommended for use by
ICCVAM pending receipt of additional information, which the commenter supported,and using alternative decision criteria. The commenter further noted that ICCVAM
qualified their acceptance and recommended a weight-of-evidence approach. The
commenter indicated that while it is usually good scientific practice to evaluate any
test method results in a weight-of-evidence manner, qualifications such as thesechallenged the recommendations and gave incentive to conduct more testing, when in
reality the method evaluated had acceptable performance and should simply berecommended.
•

The January 2008 draft ICCVAM recommendations for the LLNA: BrdU-ELISA
indicated that the test method may be useful for identifying substances as potential
skin sensitizers and nonsensitizers but recommended that more data and information
were needed before final recommendations could be made. The January 2008 draft
ICCVAM recommendations did not recommend using a weight-of-evidence
approach to hazard classification.

2. Another commenter agreed with the January 2008 draft ICCVAM recommendationthat more information and data were needed for the LLNA: BrdU-ELISA in order to
conduct a meaningful assessment of the procedure’s performance relative to the
traditional LLNA. The commenter further agreed with the ICCVAM
recommendation that it was important to have information regarding theinterlaboratory performance of the assay. The commenter also had a suggestion
regarding Table 6-2 of the January 2008 draft BRD. Since an alternative SI cutoff for
the LLNA: BrdU-ELISA was identified (i.e., SI ≥ 1.3) a comparison of
LLNA: BrdU-ELISA EC1.3 values to traditional LLNA EC3 values would be
helpful.
•A comparison of data for the alternative SI values is included in the final ICCVAM
BRD (see Appendix C).

Two oral comments were relevant to the LLNA: BrdU-ELISA.

1. One commenter agreed with ICCVAM that the LLNA: BrdU-ELISA and the LLNA:
DA should be evaluated separately from one another because they have different
treatment schedules. The tests have very little similarity, other than using CBA miceand measuring lymphocyte proliferation.
2. Another commenter explained that the rationale for selection of the CBA/JN strain ofmice for the LLNA: BrdU-ELISA was that the sensitivity of the strain to pbenzoquinone was greater than that of the other two strains tested (i.e., BALB/cAnN
and CD-1).

4.2.4

Public Comments in Response to 73 FR 25754 (May 7, 2008): Meeting of the
Scientific Advisory Committee on Alternative Toxicological Methods(SACATM)

26B

NICEATM announced the SACATM meeting and requested written and public oral comments on
the agenda topics. One public comment was received in response to this FR notice. The
commenter made a general comment that the members of SACATM do not represent a crosssection of the American public.
•

4.2.5

The SACATM charter indicates that the Committee shall consist of 15 members,
including the Chair. Voting members shall be appointed by the Director, National
Institute of Environmental Health Sciences (NIEHS), and include representatives
from an academic institution, a State government agency, an international regulatory
body, or any corporation developing or marketing new or revised or alternative test
methodologies, including contract laboratories. Knowledgeable representatives frompublic health, environmental communities, or organizations using new or alternative
test methodologies may be included as appropriate. There shall be at least one
knowledgeable representative having a history of expertise, development, or
evaluation of new or revised or alternative test methods from each of the following
categories: (1) personal care, pharmaceutical, industrial chemicals, or agriculturalindustry; (2) any other industry that is regulated by one of the Federal agencies on
ICCVAM; and (3) a national animal protection organization established under
section 501(c)(3) of the Internal Revenue Code of 1986. The Director, NIEHS, shallselect the Chair from among the appointed members of SACATM.

Public Comments in Response to 73 FR 29136 (May 20, 2008): Peer Review
Panel Report on the Validation Status of New Versions and Applications of
the Murine Local Lymph Node Assay (LLNA): A Test Method for Assessing
the Allergic Contact Dermatitis Potential of Chemicals and Products: Noticeof Availability and Request for Public Comments
27B

NICEATM requested submission of written public comments on the Independent Scientific Peer
Review Panel Assessment. No public comments were received in response to this FR notice.

4.2.6

Public and SACATM Comments: SACATM Meeting on June 18-19, 2008

28B

The June 18-19, 2008, SACATM meeting included a discussion of the ICCVAM review of the
LLNA test method (see Appendix E3).
There were no public comments specific to the LLNA: BrdU-ELISA.
Regarding the LLNA: BrdU-ELISA, one SACATM member indicated that the LLNA BrdUELISA had potential based on an accuracy of 83% (19/23) but a detailed protocol had not been
provided and it was premature to make judgments.

The January 2008 draft ICCVAM recommendations included a statement that a sufficiently
detailed protocol of the test method, including a defined and adequately justified decision
criterion for distinguishing between sensitizers and nonsensitizers, was required. NICEATM
subsequently obtained the detailed protocol, which was included in the revised draft BRD thatwas evaluated by the Panel in April 2009.

4.2.7

Public Comments in Response to 74 FR 8974 (February 27, 2009):
Announcement of a Second Meeting of the Independent Scientific Peer
Review Panel on the Murine Local Lymph Node Assay; Availability of Draft
Background Review Documents (BRD); Request for Comments
29B

NICEATM requested public comments on the revised draft BRDs, revised draft ICCVAM test
recommendations, revised draft test method protocols, and revised draft LLNA performancestandards for the second international independent scientific peer review panel meeting toevaluate modifications and new applications for the LLNA. NICEATM received three comments
in response to this FR notice: one written comment, and two oral comments offered at the Panelmeeting.
1. There was a general comment expressing concern that the extensive time andresources that ICCVAM has devoted to this evaluation has detracted from focus onpromising in vitro methods with potential to have a much greater impact on animal
use.
•

ICCVAM considers that the evaluations conducted to date have significant potential
to further reduce and refine animal use, particularly where the use of the LLNA isprecluded due to restrictions associated with the use of radioactivity. ICCVAM is
also committed to identifying in vitro models and non-animal approaches forassessing ACD and is engaged with ECVAM and JaCVAM in the development ofvalidation studies for such methods.

The commenter further made one written comment relevant to the LLNA: BrdU-ELISA.
1. The commenter supported the revised draft ICCVAM recommendation that the
LLNA: BrdU-ELISA can be used for ACD testing with specific defined limitationsin the decision criteria. That is, that substances falling within the intermediate SI
would be subjected to an integrated decision strategy in conjunction with all otheravailable information (e.g., dose response information, statistical analyses of treated
vs. control animals, peptide reactivity, molecular weight, results from related
chemicals, other testing data). While the commenter offered general support for thisuse, they emphasized that it should be made clear that “other testing data” refers to
retrospective analyses rather than initiation of additional tests in animals.
•

ICCVAM agrees that additional animal tests should be avoided whenever possible.
The intermediate SI range was discarded because it was irrelevant for ICCVAM’s
final recommendation to use a single decision criterion, SI ≥ 1.6, to classify
sensitizers. However, ICCVAM recommends that borderline positive results (i.e., SI
values between 1.6 and 1.9) should be evaluated with other available information(e.g., dose-response information, evidence of systemic toxicity or excessive local
irritation, statistical comparison of treated vs. vehicle control groups [whereappropriate], peptide reactivity, molecular weight, results from related substances,other testing data) to confirm that such results are positive.

2. The commenter further noted that the Panel recommended that the LLNA: BrdUELISA and the two other nonradioactive methods should be evaluated for theirability to assess mixtures, metals, and aqueous solutions concurrently with theassessment of these substances in the traditional LLNA. The commenter viewed thatsince the only difference between these methods and the traditional LLNA is themethod of detection, it is unlikely that there will be any differences in theapplicability of these methods and the traditional LLNA with regard to mixtures,
metals and aqueous solutions. Therefore, it would be highly inappropriate to performthese redundant studies, especially since there are no available data for comparison.
•As outlined in the test method recommendations, ICCVAM considers theapplicability domain for the nonradioactive LLNA methods to be the same as the
traditional LLNA unless there are properties associated with a class of materials that
may interfere with the accuracy of the LLNA: BrdU-ELISA.

One oral comment was relevant to the LLNA: BrdU-ELISA.
1. One commenter stated that the nonradiolabeled LLNA methods should not be held toa higher standard than the traditional LLNA.
•

4.2.8

ICCVAM evaluated the LLNA: BrdU-ELISA test method based on the applicable
criteria for validation and acceptance of toxicological test methods in the ICCVAM
submission guidelines (ICCVAM 2003). ICCVAM is committed to ensuring that new
methods are equivalent to or better than the currently accepted toxicological test
methods in order to protect public health.

Public Comments in Response to 74 FR 19562 (April 29, 2009): Meeting of
the Scientific Advisory Committee on Alternative Toxicological Methods(SACATM)
30B

NICEATM announced the SACATM meeting and requested written and public oral comment on
the agenda topics. No public comments were received in response to this FR notice.

4.2.9

Public Comments in Response to 74 FR 26242 (June 1, 2009): Independent
Scientific Peer Review Panel Report: Updated Validation Status of New
Versions and Applications of the Murine Local Lymph Node Assay: A Test
Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals
and Products: Notice of Availability and Request for Public Comments
31B

NICEATM requested submission of written public comments on the Independent Scientific Peer
Review Panel Assessment. One comment was received in response to this FR notice.
The commenter made one comment relevant to the LLNA: BrdU-ELISA.
1. The commenter did not consider the nonradioactive LLNA methods to provide
significant advantages to the traditional LLNA.
•

The ICCVAM recommendations for the nonradioactive test methods state that the
proposed nonradioactive modifications to the LLNA test method protocol havesignificant potential to further reduce and refine animal use, given that they will
likely increase the use of the LLNA instead of GP test methods where radioactivity is
prohibited.

The commenter also indicated that the number of animals used in the LLNA: BrdU-ELISA waseight animals per dose group and for ethical reasons the LLNA: BrdU-ELISA might be avoided.

•The commenter misunderstood the number of animals required by the LLNA: BrdUELISA. The ICCVAM-recommended protocol for the LLNA: BrdU-ELISA indicatesthat four animals per dose group are recommended.

The commenter further indicated that the justification for replacing the GP is not provided for the
LLNA: BrdU-ELISA and that it should be mentioned.
•

As indicated in Section 10.0 of the final ICCVAM BRD (Appendix C), the LLNA:
BrdU-ELISA evaluates only the induction phase of skin sensitization and therefore
discomfort to animals associated with the elicitation phase is eliminated.
Additionally, the LLNA: BrdU-ELISA test method protocol requires fewer mice pertreatment group (a minimum of four animals per group) than either of the GP tests(10-20 animals/group for the Buehler test and 5-10 animals/group for GPMT).

4.2.10 Public and SACATM Comments: SACATM Meeting on June 25-26, 200932B

The June 25-26, 2009, SACATM meeting included a discussion of the ICCVAM review of the
LLNA test method (see Appendix E4).
There were no public comments specific to the LLNA: BrdU-ELISA.
In general, SACATM was supportive of the Panel report. However, there was general concern
regarding the potential for overlabeling substances that may occur by using LLNA test results.
They emphasized the need for developing non-animal test methods for identifying potential skinsensitizers.
One SACATM member commented that many laboratories had moved away from using the
LLNA because it used radioactivity. Therefore, the option of LLNA test method protocols that do
not use radioactivity would likely increase use of the LLNA.
Regarding the LLNA: BrdU-ELISA, another SACATM member indicated that the use of two SI
decision criteria in the LLNA: BrdU-ELISA (i.e., one for determining sensitizers and one fordetermining nonsensitizers) could potentially place many compounds in the range of uncertainty(i.e., the range in which maximum SI results were between the SI decision criteria for sensitizers
and nonsensitizers), so the decision criteria should be reassessed as more data are obtained.
•

The final ICCVAM recommendations state that a single decision criterion of SI ≥ 1.6
be used to classify substances as potential sensitizers since there were no false
negatives in the current validation database, relative to the traditional LLNA, when
this criterion is used. However, using an SI ≥ 1.6 as the decision criterion results in a
false positive rate of 18% (2/11) compared to the traditional LLNA. Since the two
false positive substances in the LLNA: BrdU-ELISA produced SI values between 1.6
and 1.9, users may want to consider additional information (e.g., dose-response
information, evidence of systemic toxicity and/or excessive local skin irritation,
statistical comparison of treated vs. vehicle control groups [where appropriate],
peptide reactivity, molecular weight, results from related substances, or other testingdata) to confirm that such results in the SI range are positive.

5.0

References

4B

Buehler EV. 1965. Delayed contact hypersensitivity in the guinea pig. Arch Dermatol 91:171177.
Dean J, Twerdok L, Tice R, Sailstad D, Hattan D, Stokes WS. 2001. ICCVAM evaluation of themurine local lymph node assay. Conclusions and recommendations of an independent scientificpeer review panel. Regul Toxicol Pharmacol 34:258-273.
ESAC. 2007. Statement on the Reduced Local Lymph Node Assay (rLLNA). European
Commission Directorate General, Joint Research Centre, Institute for Health and Consumer
Protection, European Centre for the Validation of Alternative Methods. Available at:
http://ecvam.jrc.it/ft_doc/ESAC26_statement_rLLNA_20070525-1.pdf.
Gerberick GF, Ryan CA, Kern PS, Dearman RJ, Kimber I, Patlewicz GY, et al. 2004. A chemical
dataset for evaluation of alternative approaches to skin-sensitization testing. Contact Dermatitis
50:274-288.
Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP. 2007.
Quantification of chemical peptide reactivity for screening contact allergens: a classification treemodel approach. Toxicol Sci 97:417-427.
Haneke KE, Tice RR, Carson BL, Margolin BH, Stokes WS. 2001. ICCVAM evaluation of themurine local lymph node assay: III. Data analyses completed by the National Toxicology
Program Interagency Center for the Evaluation of Alternative Toxicological Methods. Regul
Toxicol Pharmacol 34:274-286.
Hutchings CV, Shum KW, Gawkrodger DJ. 2001. Occupational contact dermatitis has anappreciable impact on quality of life. Contact Dermatitis 45:17-20.
ICCVAM 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemical/Compounds. NIH Publication No. 99-4494.
Research Triangle Park, NC:National Toxicology Program. Available:
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf .
H

H

ICCVAM Authorization Act. 2000. Public Law 106-545, 42 U.S.C. 285l-3.
ICCVAM. 2009a. Recommended Performance Standards: Murine Local Lymph Node Assay.
NIH Publication No. 09-7357. Research Triangle Park, NC:National Institute of Environmental
Health Sciences. Available at http://iccvam.niehs.nih.gov/methods/immunotox/llna_PerfStds.htm.
ICCVAM. 2009b. ICCVAM Test Method Evaluation Report. The Reduced Murine Local Lymph
Node Assay: An Alternative Test Method Using Fewer Animals to Assess the Allergic Contact
Dermatitis of Potential of Chemicals and Products. NIH Publication No. 09-6439. Research
Triangle Park, NC:National Institute of Environmental Health Sciences. Available at
http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-LD/TMER.htm.
Kimber I, Dearman RJ, Betts CJ, Gerberick GF, Ryan CA, Kern PS, et al. 2006. The local lymph
node assay and skin sensitization: a cut-down screen to reduce animal requirements? Contact
Dermatitis 54:181-185.
Kojima H, Takeyoshi M, Omori T, Sozu, T, Arima K, Idehara K, et al. 2008. Inter-laboratory
validation study on LLNA-BrdU. Toxicologist 102:216.
Magnusson B, Kligman AM. 1970. Allergic Contact Dermatitis in the Guinea Pig. Springfield,
IL:Charles C. Thomas.

OECD. 2002. Test Guideline 429. Skin Sensitisation: Local Lymph Node Assay, adopted April
24, 2002. In: OECD Guidelines for Testing of Chemicals. Paris:OECD. Available:
http://titania.sourceoecd.org/vl=7033968/cl=16/nw=1/rpsv/cw/vhosts/oecdjournals/1607310x/v1n4/contp1-1.htm
H

Sailstad DM, Hattan D, Hill RN, Stokes WS. 2001. ICCVAM evaluation of the murine local
lymph node assay (LLNA) I: The ICCVAM review process. Regul Toxicol Pharmacol 34:249257.
Skoet R, Zachariae R, Agner T. 2003. Contact dermatitis and quality of life: a structured reviewof the literature. Br J Dermatol 149:452-456.
Takeyoshi M, Iida K, Shiraishi K, Hoshuyama S. 2005. Novel approach for classifying chemicals
according to skin sensitizing potency by non-radioisotopic modification of the local lymph node
assay. J Appl Toxicol 25:129-34.
Takeyoshi M, Iida K, Suzuki K, Yamazaki S. 2007a. Skin sensitization potency of isoeugenol andits dimers evaluated by a non-radioisotopic modification of the local lymph node assay andguinea pig maximization test. J Appl Toxicol DOI:10.1002/jat.1305 [Online 10 October 2007].
Takeyoshi M, Minobe Y, Takatsuki M. 2007b. Promising endpoint and assay performance of
non-radioisotopic local lymph node assay based on BrdU incorporation. Presentation at the 6th
World Congress on Alternatives and Animal Use in the Life Sciences. Tokyo, Japan.
Takeyoshi M, Noda S, Yamasaki K. 2004a. Differences in responsiveness of mouse strain against
p-benzoquinone as assessed by non-radioisotopic murine local lymph node assay. Exp Anim
53:171-173.
Takeyoshi M, Noda S, Yamazaki S, Kakishima H, Yamasaki K, Kimber I. 2004b. Assessment of
the skin sensitization potency of eugenol and its dimers using a non-radioisotopic modification of
the local lymph node assay. J Appl Toxicol 24:77-81.
Takeyoshi M, Noda S, Yamasaki K, Kimber I. 2006. Advantage of using CBA/N strain mice in a
non-radioisotopic modification of the local lymph node assay. J Appl Toxicol 26:5-9.
Takeyoshi M, Sawaki M, Yamasaki K, Kimber I. 2003. Assessment of statistic analysis in nonradioisotopic local lymph node assay (non-RI-LLNA) with alpha-hexylcinnamic aldehyde as an
example. Toxicology 191:259-263.
Takeyoshi M, Yamasaki K, Yakabe Y, Takatsuki M, and Kimber I. 2001. Development of nonradio isotopic endpoint of murine local lymph node assay based on 5-bromo-2'-deoxyuridine(BrdU) incorporation. Toxicology Letters 119:203-208.

This page intentionally left blank